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Stadium Judo Club

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Luke Edwards
Luke Edwards

Stalk


Sprouting angiogenesis is a dynamic process in which endothelial cells collectively migrate, shape new lumenized tubes, make new connections, and remodel the nascent network into a hierarchically branched and functionally perfused vascular bed. Endothelial cells in the nascent sprout adopt two distinct cellular phenotypes--known as tip and stalk cells--with specialized functions and gene expression patterns. VEGF and Notch signaling engage in an intricate cross talk to balance tip and stalk cell formation and to regulate directed tip cell migration and stalk cell proliferation. In this article, we summarize the current knowledge and implications of the tip/stalk cell concepts and the quantitative and dynamic integration of VEGF and Notch signaling in tip and stalk cell selection.




stalk



An HA stalk-based vaccination strategy provides broad protection from challenge with divergent H3N2 viruses. (A) Schematic representation of the vaccination strategy (the monomeric form of each antigen is shown). (B to E) Animals were vaccinated with plasmid DNA coding for cH4/3 HA and subsequently boosted with recombinant soluble cH5/3, followed by cH7/3 proteins (green triangles; n = 9 or 10 animals). Positive-control animals received inactivated vaccine containing the matched challenge strain (red circles; n = 5 animals). Prime-only animals (orange triangles; n = 5 animals) received the DNA prime followed by two irrelevant protein boosts. Naive animals (black squares; n = 5 animals) were used as additional controls for challenge. (B) Weight loss curves upon challenge with the Phil82 (H3N2) virus. (C) Kaplan-Meier survival curve upon Phil82 challenge. (D) Morbidity observed upon challenge with the X-31 (H3N2) virus. (E) Survival curves following the X-31 (H3N2) challenge. Survival of vaccinated (cH4/3DNA-cH5/3-cH7/3) versus control (cH4/3DNA-BSA-BSA) groups is highly significant for both challenge experiments (P = 0.0008 and 0.0001, respectively). (F and G) To further test the protection breadth of the vaccine against viruses that are not lethal in the mouse model, we performed lung titration experiments. Vaccinated animals (BcH7/3-cH5/3-cH4/3) and control animals (Bwt-BSA-BSA) were infected with 5 104 PFU of H3N2 variant (F) or 1 105 PFU of the human H3N2 A/Wyoming/03/03 (G). On day 3 postinfection, lungs of animals from both groups were harvested and homogenized, and the 50% tissue culture infectious dose (TCID50) was measured.


Vaccination with cHA constructs can boost preexistent titers of stalk-reactive antibodies to protective levels. (A to G) To mimic the preexisting immunity to the stalk domain present in the human population, animals were sublethaly infected with a recombinant influenza B virus that expresses cH7/3 HA. Subsequently, they were boosted with recombinant soluble cH5/3 (or full-length H3 HA for cH5/3N1-challenged animals) and then cH4/3 protein (green triangles; n = 10 animals). Positive-control animals received inactivated vaccine containing the matched challenge strain (red circles; n = 5 animals). Prime-only animals (orange triangles; n = 5 animals) received the recombinant influenza B prime and then two irrelevant protein boosts. Additional control groups were either infected with wild-type influenza B virus and then received two irrelevant protein boosts (light-green triangles; n = 5) or were naive (black squares; n = 5). (B to D) Weight loss curves following viral challenges. (B) We used a mouse-pathogenic cH5/3N1 virus which expresses the stalk domain of an HA from a recent human H3N2 isolate as the surrogate challenge strain to test efficacy against contemporary stalk domains, since modern human H3N2 isolates are not pathogenic in mice. Weight loss upon infection with the X-31 (H3N2; HA and NA from A/Hong Kong/1/68) (C) and Phil82 (H3N2) (D) viruses; (E) Kaplan-Meier survival curve following the A/cH5/3N1 challenge; (F) survival curves following the Phil82 (H3N2) challenge; (G) survival curves following the X-31 (H3N2) challenge. Statistical analysis revealed high significance for all challenges when comparing BcH7/3-cH5/3-cH4/3 and BcH7/3-BSA-BSA groups (P = 0.082, P


The elicited anti-stalk responses are cross-reactive against multiple H3N2 strains, including the most recent vaccine strain. (A to E) ELISA reactivity against the current influenza vaccine strain Vic11 (H3N2, whole virus) (A), Perth09 (H3 protein) (B), H3N8 virus (C), Phil82 virus (H3N2) (D), or X-31 virus (H3N2; expressing HA and NA from A/Hong Kong/1/68 virus) (E) of sera collected from animals vaccinated with cHA constructs (dark-green triangles, cH4/3DNA-cH5/3-cH7/3), prime-only animals (orange triangles), or naive animals (black squares). (F to H) ELISA reactivity against Vic11 H3N2 (F) or Phil82 H3N2 (G) or X-31 H3N2 (H) virus of sera collected from animals vaccinated with cHA constructs (green triangles, B/cH7/3 virus-cH5/3 protein-cH4/3 protein), prime-only animals (orange triangles), control animals that were infected with wild-type influenza B virus and then received two BSA boosts (light-green triangles), or naive animals (black squares).


The polyclonal responses elicited by the chimeric HA vaccination are directed against the stalk domain and neutralize virus infection both in vitro and in vivo. (A) An ELISA against a group 1 HA protein (H1) demonstrates that the cross-reactive responses elicited by the cHA vaccine (dark-green triangles, B/cH7/3-cH5/3-cH4/3) are not directed against conserved parts of the receptor binding site in the HA protein. (B) ELISA reactivity of nasal washes from animals vaccinated with cHA constructs (dark-green triangles, B/cH7/3-cH5/3 protein-cH4/3 protein), prime-only animals (orange triangles, B/cH7/3-irrelevant protein-irrelevant protein), vector controls (light-green squares, Bwt-irrelevant protein-irrelevant protein), and naive animals (black squares). (C) Antibody isotypes in sera from vaccinated (B/cH7/3 virus-cH5/3 protein-cH4/3 protein), naive, prime-only (B/cH7/3 virus-BSA-BSA), and vector control animals (Bwt virus-BSA-BSA). (D) Vic11 (H3) pseudotyped particle neutralization assay with sera from cHA-vaccinated animals (dark-green triangles, B/cH7/3-cH5/3-cH4/3), vector controls (light-green squares, Bwt-irrelevant protein-irrelevant protein), and naive animals (black squares). The reciprocal serum dilution is shown on the x axis. An H3 stalk-reactive monoclonal antibody (12D1) was used as positive control (red triangles), at a starting concentration of 123 μg/ml. (E) Passive transfer challenge experiment (Phil82 H3N2 virus) with sera from animals that were vaccinated (dark-green triangles, BcH7/3-cH5/3-cH4/3, n = 5 animals), vector controls (light-green squares, Bwt-BSA-BSA, n = 5 animals), naive animals (black squares, n = 5 animals), and positive-control animals (received inactivated Phil82 virus vaccine, n = 5 animals). Kaplan-Meier survival curve is shown (P


In addition to the serum IgG titers, we also assessed levels of secretory IgA on the mucosal surfaces of vaccinated mice. We detected high reactivity to Perth09 H3 HA in nasal washes collected from the group of mice that received the vaccine, whereas nasal washes from control animals did not react to the substrate (see Fig. 5B). Although mucosal anti-stalk IgA antibodies, and their ability to block viral infection, have not been yet formally characterized, we expect that they contribute to the observed protection.


The breadth of the anti-H3 HA stalk antibodies elicited by the cHA vaccination strategy extends to other members of the group 2 HAs, including the most recent Chinese H7N9 virus. (A) To ensure that anti-H7 globular-head domain antibodies are not involved in the effects observed in this series of experiments, we modified the vaccination scheme presented in Fig. 1. Animals received the same prime (DNA coding for cH4/3 HA) and first boost (cH5/3 protein), but the second boost was replaced with an H3 protein (cH4/3DNA-cH5/3-H3; green triangles, n = 10 animals). Positive-control animals received inactivated RheaH7 virus vaccine (red circles, n = 5). Prime-only animals (orange triangles, n = 5 animals) received the DNA prime followed by two irrelevant protein boosts. Naive animals (black squares, n = 5 animals) were used as additional controls. (B) Weight loss curve upon challenge with RheaH7 (H7N1) virus. (C) Kaplan-Meier survival curve. The vaccine provided good protection against mortality (P = 0.0088, cH4/3DNA-cH5/3-cH7/3 versus controls cH4/3DNA-BSA-BSA). (D, E) ELISA reactivity of sera from vaccinated animals (dark-green triangles, cH4/3DNA-cH5/3-cH7/3), control animals (orange triangles, cH4/3DNA-BSA-BSA), or naive mice (black squares) to RheaH7 virus (D) or the HA protein expressed by the recent Shanghai13 H7N9 (E) virus. (F) We infected mice that received the vaccine (BcH7/3-cH5/3-cH4/3) and controls (Bwt-BSA-BSA) with 1 105 PFU of H10N7 virus and measured a 20-fold decrease in viral TCID50 in the lungs of vaccinated mice on day 3 postinfection. (G) Sera collected from vaccinated mice cH4/3DNA-cH5/3-H3 recognize a panel of group 2 HA proteins. MDCK cells were transfected with plasmids encoding the respective HA and were fixed 16 h later with 0.5% paraformaldehyde. Reactivity was detected by immunofluorescence with sera from mice that received the vaccine. Serum collected from naive animals was used as a negative control. A mouse (FBE9 [unpublished, generated in-house]) and a human (FI6) (21) monoclonal anti-stalk antibody were used as positive controls.


Humans are exposed to influenza viruses multiple times throughout their lifetime and therefore are likely to have preexisting memory B cells with specificities in the stalk domain. We wanted to mimic this situation in the mouse model and see if we could efficiently boost preexisting titers of stalk-reactive antibodies. Mice were preexposed to the H3 stalk domain by being sublethally infected with a recombinant influenza B virus expressing the H3 stalk domain in combination with an irrelevant globular head domain. Upon subsequent vaccination with cHA constructs containing the same stalk domain but different heads, the levels of stalk-reactive antibodies were efficiently boosted and protected mice from challenge with a panel of H3N2 influenza virus strains spanning from 1968 to 2009. Serum from vaccinated mice showed good reactivity to a wide range of H3N2 virus substrates as well as an H7N1 virus and an H7N9 HA protein substrate. Vaccinated animals also showed reduced lung titers after infection with H3N2v, H3N8, and H10N7 infection. Furthermore, the serum showed neutralizing activity and could protect mice in a passive transfer challenge experiment. These findings shed light on the mechanism of neutralization elicited by this vaccination strategy and suggest that an antibody-mediated mechanism, likely based on virus neutralization, is mediating protection. A contribution by CD8+ and CD4+ T cells to protection cannot be ruled out at this point, although transfer of serum alone was sufficient to protect from challenge. Enhanced pathogenicity induced by nonneutralizing cross-reactive anti-influenza antibodies has been proposed as a possible reason for the high pathogenicity of the novel Chinese H7N9 virus in the elderly (44). We did not observe any enhanced pathogenicity in cHA-vaccinated animals that had high titers of cross-neutralizing antibodies. In fact the animals were protected from morbidity and mortality and the virus was cleared faster. 350c69d7ab


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