Broadly protective adenovirus-based multivalent vaccines against highly pathogenic avian influenza viruses for pandemic preparedness.

Recurrent outbreaks of H5, H7 and H9 avian influenza viruses in domestic poultry accompanied by their occasional transmission to humans have highlighted the public health threat posed by these viruses. Newer vaccine approaches for pandemic preparedness against these viruses are needed, given the limitations of vaccines currently approved for H5N1 viruses in terms of their production timelines and the ability to induce protective immune responses in the absence of adjuvants. In this study, we evaluated the feasibility of an adenovirus (AdV)-based multivalent vaccine approach for pandemic preparedness against H5, H7 and H9 avian influenza viruses in a mouse model. Replication-defective AdV vectors expressing hemagglutinin (HA) from different subtypes and nucleoprotein (NP) from one subtype induced high levels of humoral and cellular immune responses and conferred protection against virus replication following challenge with H5, H7 and H9 avian influenza virus subtypes. Inclusion of HA from the 2009 H1N1 pandemic virus in the vaccine formulation further broadened the vaccine coverage. Significantly high levels of HA stalk-specific antibodies were observed following immunization with the multivalent vaccine. Inclusion of NP into the multivalent HA vaccine formulation resulted in the induction of CD8 T cell responses. These results suggest that a multivalent vaccine strategy may provide reasonable protection in the event of a pandemic caused by H5, H7, or H9 avian influenza virus before a strain-matched vaccine can be produced

Virus neutralization antibody titers and virus lung titers in mice vaccinated with AdV vector-based vaccines and challenged with influenza viruses from H1, H5, H7, H9, and H3 subtypes.

<p>Mouse groups were immunized with H1HA, H5b+H7H9b+H1HA, or ΔE1E3 twice at four week intervals. Four weeks post-booster inoculation, serum samples were obtained from all animals for determining neutralizing antibody titers (VN) by virus microneutralization assay. Four weeks after the last immunization, mice (5 animals/group) were challenged with one of the following viruses: Egypt/08, TK/VA, G1/99, pH1N1, or X-31. Three days after challenge, the animals were euthanized, and the virus lung titers (VLT) were determined as described under Materials and Methods<b>.</b> Antibody titers are shown as geometric mean values and the virus titers are shown as a mean Log₁₀ TCID₅₀<b>±</b> standard deviation <b>(</b>SD). The detection limit of the lung viral titer was 0.5 Log₁₀ TCID₅₀/ml. H1HA, HAd-H1HA; H5b+H7H9b+H1HA, HAd-1203HA-05HA+HAd-H7HA-H9HA+HAd-H1HA; ΔE1E3, HAd-ΔE1E3; pH1N1, A/California/08/2009 (H1N1); Egypt/08, A/Egypt/3300-NAMRU3/2008 (H5N1)-PR8-IDCDC-RG13; TK/VA, A/turkey/Virginia/2002 (H7N2)-PR8-IBCDC-5; G1/99, A/Hong Kong/1073/1999 (H9N2); X-31, A/Aichi/2/1968 (H3N2)-PR8.</p

Induction of HA518 epitope pentamer-specific CD8+ T cells and ELISpot measurements of IFN-γ expression in spleen cells of vaccinated mice.

<p>Mice (7 animals/group) were immunized with 1203HA, 05HA, H5b, H7HA, H9HA, H7H9b, H5b+H7H9b or ΔE1E3 twice at four-week intervals. At four weeks after booster inoculation, the animals were euthanized, and the spleens were collected for the evaluation of HA-specific CMI responses using HA518 epitope-specific pentamer staining (a) and IFN-γ-ELISpot assay (b) as described under Materials and Methods. The data represent mean± standard deviation (SD) from 7 animals/group. ***; <i>P</i>≤0.005. 1203HA, HAd-1203HA; 05HA, HAd-05HA; H5b, HAd-1203HA-05HA; H7HA, HAd-H7HA; H9HA, HAd-H9HA; H7H9b, HAd-H7HA-H9HA; H5b+H7H9b; HAd-1203HA-05HA+HAd-H7HA-H9HA; ΔE1E3, HAd-ΔE1E3.</p

Virus lung titers in vaccinated mice challenged with influenza viruses from H5, H7, H9, H1 and H3 subtypes.

<p>Mouse groups were immunized with H5b, H7H9b, H5b+H7H9, H5b+H7H9b+H1HA, H5b+NP, H7H9b+NP, H5b+H7H9b+NP, H5b+H7H9b+H1HA+ NP, or ΔE1E3 twice at 4 week intervals. Four weeks post-booster inoculation, the mice (5 animals/group) were challenged with one of the following viruses: Egypt/08, TK/VA, G1/99, pH1N1, or X-31. Three days after challenge, the animals were euthanized, and the lung virus titers were determined as described under Materials and Methods. The data are shown as the mean Log₁₀ TCID₅₀ titers ± SD. The detection limit of the lung viral titer was <0.5 Log₁₀ TCID₅₀/ml (indicated as <0.50).</p>*<p>, <i>P</i>≤0.050 and **, <i>P</i><0.010 compared to ΔE1E3 control. H5b, HAd-1203HA-05HA; H7H9b, HAd-H7HA-H9HA; H5b+H7H9b; HAd-1203HA-05HA+HAd-H7HA-H9HA; H5b+H7H9b+H1HA, HAd-1203HA-05HA+HAd-H7HA-H9HA+HAd-H1HA; H5b+NP, HAd-1203HA-05HA+HAd-NP; H7H9b+NP, HAd-H7HA-H9HA+HAd-NP; H5b+H7H9b+NP; HAd-1203HA-05HA+HAd-H7HA-H9HA+HAd-NP; H5b+H7H9b+H1HA+NP; HAd-1203HA-05HA+HAd-H7HA-H9HA+HAd-H1HA+HAd-NP; ΔE1E3, HAd-ΔE1E3; Egypt/08, A/Egypt/3300-NAMRU3/2008 (H5N1)-PR8-IDCDC-RG13; TK/VA, A/turkey/Virginia/2002 (H7N2)-PR8-IBCDC-5; G1/99, A/Hong Kong/1073/1999 (H9N2); pH1N1, A/California/08/2009 (H1N1); X-31, A/Aichi/2/1968 (H3N2)-PR8.</p

HA518 and NP147 epitope-specific CD8+ T cells and ELISpot measurements of IFN-γ expression in spleen cells of vaccinated mice.

<p>Mice (7 animals/group) were immunized with H5b, H7H9b, H5b+H7H9b, H5b+NP, H7H9b+NP, H5b+H7H9b+NP or ΔE1E3 twice at four-week intervals. At four weeks after booster inoculation, the animals were euthanized, and the spleens were collected for the evaluation of HA-specific and NP-specific CMI responses using pentamer staining (a) and IFN-γ-ELISpot assay (b) as described under Materials and Methods. The data represent mean± standard deviation (SD) from 7 animals/group. **; <i>P</i><0.010 and ***; <i>P</i>≤0.005 compared to ΔE1E3. H5b, HAd-1203HA-05HA; H7H9b, HAd-H7HA-H9HA; H5b+H7H9b; HAd-1203HA-05HA+HAd-H7HA-H9HA; H5b+NP, HAd-1203HA-05HA+HAd-NP; H7H9b+NP, HAd-H7HA-H9HA+HAd-NP; H5b+H7H9b+NP, HAd-1203HA-05HA+HAd-H7HA-H9HA+HAd-NP; ΔE1E3, HAd-ΔE1E3.</p

Induction of antibodies against the HA stalk region following immunization with AdV vector-based vaccines.

<p>Mice (7 animals/group) were immunized with 1203HA, 05HA, H5b, H7HA, H9HA, H7H9b, H1HA, H5b+H7H9b, or ΔE1E3 twice at four-week intervals. Four weeks post-booster inoculation, serum samples were obtained for determining ELISA antibody titers against peptides P1 (a and b) and P2 (c and d) as described under Materials and Methods. The data represent mean titers ± standard deviation (SD) from 7 animals per group. 1203HA, HAd-1203HA; 05HA, HAd-05HA; H5b, HAd-1203HA-05HA; H7HA, HAd-H7HA; H9HA, HAd-H9HA; H7H9b, HAd-H7HA-H9HA; H1HA, HAd-H1HA; H5b+H7H9b; HAd-1203HA-05HA+HAd-H7HA-H9HA; ΔE1E3, HAd-ΔE1E3.</p

Protective efficacy of AdV vector-based vaccines against challenge with influenza viruses from H5, H7, and H9 subtypes.

<p>Mouse groups were immunized with 1203HA, 05HA, H5b, H7HA, H9HA, H7H9b, H5b+H7H9b or ΔE1E3 twice at four-week intervals. Four weeks post-booster inoculation, the mice (5 animals/group) were challenged with one of the following viruses: Egypt/08 (a), TK/VA (b), or G1/99 (c). Three days after challenge, the animals were euthanized, and the lung virus titers were determined as described under Materials and Methods. The data represent the mean virus titers ± standard deviation (SD). The detection limit of the lung viral titer was 0.5 Log₁₀ TCID₅₀/ml. 1203HA, HAd-1203HA; 05HA, HAd-05HA; H5b, HAd-1203HA-05HA; H7HA, HAd-H7HA; H9HA, HAd-H9HA; H7H9b, HAd-H7HA-H9HA; H5b+H7H9b; HAd-1203HA-05HA+HAd-H7HA-H9HA; ΔE1E3, HAd-ΔE1E3; Egypt/08, A/Egypt/3300-NAMRU3/2008 (H5N1)-PR8-IDCDC-RG13; TK/VA, A/turkey/Virginia/2002 (H7N2)-PR8-IBCDC-5; G1/99, A/Hong Kong/1073/1999 (H9N2).</p

Platelet factor 4 mediates inflammation in experimental cerebral malaria

Cerebral malaria (CM) is a major complication of Plasmodium falciparum infection in children. The pathogenesis of CM involves vascular inflammation, immune stimulation, and obstruction of cerebral capillaries. Platelets have a prominent role in both immune responses and vascular obstruction. We now demonstrate that the platelet-derived chemokine, platelet factor 4 (PF4)/CXCL4, promotes the development of experimental cerebral malaria (ECM). Plasmodium-infected red blood cells (RBCs) activated platelets independently of vascular effects, resulting in increased plasma PF4. PF4 or chemokine receptor CXCR3 null mice had less severe ECM, including decreased T cell recruitment to the brain, and platelet depletion or aspirin treatment reduced the development of ECM. We conclude that Plasmodium-infected RBCs can directly activate platelets, and platelet-derived PF4 then contributes to immune activation and T cell trafficking as part of the pathogenesis of ECM