Wild Type and Mutant 2009 Pandemic Influenza A (H1N1) Viruses Cause More Severe Disease and Higher Mortality in Pregnant BALB/c Mice

BACKGROUND: Pregnant women infected by the pandemic influenza A (H1N1) 2009 virus had more severe disease and higher mortality but its pathogenesis is still unclear. PRINCIPAL FINDINGS: We showed that higher mortality, more severe pneumonitis, higher pulmonary viral load, lower peripheral blood T lymphocytes and antibody responses, higher levels of proinflammatory cytokines and chemokines, and worse fetal development occurred in pregnant mice than non-pregnant controls infected by either wild type (clinical isolate) or mouse-adapted mutant virus with D222G substitution in hemagglutinin. These disease-associated changes and the lower respiratory tract involvement were worse in pregnant mice challenged by mutant virus. Though human placental origin JEG-3 cell line could be infected and proinflammatory cytokines or chemokines were elevated in amniotic fluid of some mice, no placental or fetal involvement by virus were detected by culture, real-time reverse transcription polymerase chain reaction or histopathological changes. Dual immunofluorescent staining of viral nucleoprotein and type II alveolar cell marker SP-C protein suggested that the majority of infected alveolar epithelial cells were type II pneumocytes. CONCLUSION: The adverse effect of this pandemic virus on maternal and fetal outcome is largely related to the severe pulmonary disease and the indirect effect of inflammatory cytokine spillover into the systemic circulation

A Recombinant Vaccine of H5N1 HA1 Fused with Foldon and Human IgG Fc Induced Complete Cross-Clade Protection against Divergent H5N1 Viruses

Development of effective vaccines to prevent influenza, particularly highly pathogenic avian influenza (HPAI) caused by influenza A virus (IAV) subtype H5N1, is a challenging goal. In this study, we designed and constructed two recombinant influenza vaccine candidates by fusing hemagglutinin 1 (HA1) fragment of A/Anhui/1/2005(H5N1) to either Fc of human IgG (HA1-Fc) or foldon plus Fc (HA1-Fdc), and evaluated their immune responses and cross-protection against divergent strains of H5N1 virus. Results showed that these two recombinant vaccines induced strong immune responses in the vaccinated mice, which specifically reacted with HA1 proteins and an inactivated heterologous H5N1 virus. Both proteins were able to cross-neutralize infections by one homologous strain (clade 2.3) and four heterologous strains belonging to clades 0, 1, and 2.2 of H5N1 pseudoviruses as well as three heterologous strains (clades 0, 1, and 2.3.4) of H5N1 live virus. Importantly, immunization with these two vaccine candidates, especially HA1-Fdc, provided complete cross-clade protection against high-dose lethal challenge of different strains of H5N1 virus covering clade 0, 1, and 2.3.4 in the tested mouse model. This study suggests that the recombinant fusion proteins, particularly HA1-Fdc, could be developed into an efficacious universal H5N1 influenza vaccine, providing cross-protection against infections by divergent strains of highly pathogenic H5N1 virus

Viral challenge and sample collections in pregnant and non-pregnant mice.

<p>Four groups of the mice (12-13 mice/group) were intranasally inoculated with wild type or mutant viruses. Four groups of mice were sampled at day 3 and 6 post-infection, respectively. The experiment was duplicated. This table showed the total numbers of mice in these two experiments.</p

Effect of wild type and mutant pandemic virus infection on the development of mouse fetus.

<p>Effect of wild type and mutant pandemic virus infection on the development of mouse fetus.</p

Viral infection and replication in lungs.

<p>A. Virus titers in lung homogenates collected from mice infected with wild type (WT) or mutant (Mut) virus on indicated days post-infection were measured by plaque forming assay. ** indicates significant differences (<i>P</i><0.001) between pregnant and non-pregnant mice. B. Viral RNA copies in the same lung homogenates were also determined by real-time RT-PCR. * indicates significant differences (<i>P</i><0.001) between pregnant and non-pregnant mice. C. Immunohistochemical staining for influenza viral nucleoprotein (NP) protein in lung sections of infected mice. Representative images of lung sections from wild type virus infected bronchial epithelium of non-pregnant (a) and pregnant (b) mice, and mutant virus infected alveoli of non-pregnant (c) and pregnant (d) mice. Original magnification: 100×. The NP positive cells were morphologically identified to be type I alveolar cells (few) indicated by arrow heads and type II alveolar cells (majority) indicated by arrows (e). Original magnification: 200×. Dual-labeling of mouse lung type II alveolar cells with anti-NP antibody conjugated with FITC and anti-SP-C antibody conjugated with Texas red further confirmed that the virus mainly infected type II alveolar cells (f). Arrows indicates NP and SP-C double positive alveolar cells. Original magnification: 400×.</p

Pathological and immunological changes in placenta and fetus.

<p>A. Placentas collected from pregnant mice infected with mutant (a & b) or wild type (c & d) virus at day 3 post-infection were examined after H&E staining. a, ** indicated large area of necrosis, c, * indicated two smaller foci of necrosis in the junctional zone of the placenta. Arrows in b &d indicated apoptotic bodies and nuclear debris in the trophoblast. e & f are placentas from uninfected control mice showing no significant pathological changes. Magnification of a, c, e & f 200x and b & d 400x. B. Representative sections from fetal lung (a), myocardium (c) and liver (e) tissues from mutant virus infected mice but not obvious in wild type virus infected mice (b, d & f). Original magnification: 200×. C. Levels of indicated cytokines and chemokines in amniotic fluids collected from mice infected with wild type (WT) or mutant (Mut) virus and uninfected mice (Ctl) were detected by ELISA. * indicates significant differences (<i>P</i><0.05) between pregnant mice infected with wild type and mutant viruses. IL-1β: interleukin-1β, IL-6: interleukin-6, IFN-γ: interferon-γ, MIP-1α: macrophage inflammatory protein 1α, MIP-2: macrophage inflammatory protein 2; TNF-α: tumor necrosis factor-α.</p

Virus-turkey erythrocyte receptor binding avidity assay.

<p>Influenza virus cellular receptor binding affinity was tested with turkey erythrocytes pre-treated with <i>Vibrio cholerae</i> neuraminidase which mainly removes 2, 3-linked sialic acids as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0013757#s2" target="_blank">Materials and Methods</a>. A) Illustration of assay for receptor binding avidity. B) Receptor binding avidity of mut and wt viruses. Data are representative of three independent experiments. Data was analyzed by one-way ANOVA using the mean square in the ANOVA to estimate of variability. P-value<0.001. PR8, A/Puerto Rico/8/34; Mut, mutant virus; WT, wild type virus.</p

Survival rates of infected mice.

<p>Pregnant and non-pregnant BALB/c mice infected with wild type (WT) and mutant (Mut) viruses and their survivals were recorded in indicated days. ** indicates significant difference (<i>P</i><0.001) between pregnant and non-pregnant mice.</p