Results: 4

1.
Figure 4

Figure 4. Viral loads and morbidity following A/California/07/2009 challenge in ferrets.. From: Properly Folded Bacterially Expressed H1N1 Hemagglutinin Globular Head and Ectodomain Vaccines Protect Ferrets against H1N1 Pandemic Influenza Virus.

(A) Viral replication of influenza A/California/07/2009 in nasal washes following intranasal challenge. Average pfu of virus from the nasal washes of each group (4 ferrets per group) on days 1, 3, and 5 post challenges. (B) Change in body temperature and (C) percent body weight.

Surender Khurana, et al. PLoS One. 2010;5(7):e11548.
2.
Figure 3

Figure 3. Hemagglutination-inhibition (HAI) titers in ferrets.. From: Properly Folded Bacterially Expressed H1N1 Hemagglutinin Globular Head and Ectodomain Vaccines Protect Ferrets against H1N1 Pandemic Influenza Virus.

HAI antibody in ferrets (n = 4 per group) vaccinated with either 30 µg or 7.5 µg of influenza H1N1 HA1 or HA. Blood was collected at day 35 (post-dose 2). HAI responses were assessed against A/California/07/2009. Bars indicate geometric mean titer (GMT). The titer from each individual ferret is indicated by symbol. *p = 0.05 HA (1–480) low dose vs. HA (1–480) high dose.

Surender Khurana, et al. PLoS One. 2010;5(7):e11548.
3.
Figure 2

Figure 2. Development of neutralizing and anti-HA binding antibodies following wt H1N1 (A/California/7/2009) infection in ferrets & post-H1N1 vaccination (inactivated vaccine) in humans.. From: Properly Folded Bacterially Expressed H1N1 Hemagglutinin Globular Head and Ectodomain Vaccines Protect Ferrets against H1N1 Pandemic Influenza Virus.

(A) Microneutralization of H1N1 A/California/2009 virus with post-H1N1-infected ferret samples. End-point titers (mean of three replicates) using post-infection sera from multiple ferrets at each time point in a microneutralization assay performed with A/California/07/2009 (X-179A). For day 21, sera of ten animals were pooled. Each dot in other time-points represents an individual H1N1 infected ferret. (B–D) Antibody kinetics following H1N1 challenge in ferrets. Steady-state equilibrium analysis of post-H1N1 infected ferret sera or pre- & post-H1N1 vaccinated human sera to mammalian H1N1 HA0 (Immune Technologies, NY) and properly folded bacterially expressed H1N1 HA1 (1–330) or H1N1 HA (1–480) fragment were measured using SPR. Ten-fold diluted individual post-infection sera from each time point, were injected simultaneously onto recombinant mammalian H1N1 HA0 in (B) and properly folded bacterially expressed H1N1 HA1 (1–330) in (C) or H1N1 HA (1–480) in (D), immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of peptide. SPR binding of pre-vaccine and post-H1N1 vaccination sera from two individuals with different neutralizing antibody titers (in parenthesis) is shown with recombinant mammalian H1N1 HA0 in (E) and properly folded bacterially expressed H1N1 HA1 (1–330) in (F) or H1N1 HA (1–480) in (G). Binding was recorded using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA).

Surender Khurana, et al. PLoS One. 2010;5(7):e11548.
4.
Figure 1

Figure 1. Biochemical and functional characterization of bacterially expressed and purified H1N1 HA proteins.. From: Properly Folded Bacterially Expressed H1N1 Hemagglutinin Globular Head and Ectodomain Vaccines Protect Ferrets against H1N1 Pandemic Influenza Virus.

(A) Purified E. coli derived HA proteins were analyzed by SDS-PAGE. DNA encoding HA1 (1–330) and HA (1–480) from HA gene segment of A/California/07/2009 (H1N1) generated from egg-grown virus were used for cloning in a T7 promoter based expression vector (pSK) where the desired polypeptide can be expressed as fusion protein with His6 tag at the C-terminus. The proteins were expressed, denatured and refolded under controlled redox conditions and purified using His-Trap fast flow chromatography to >90% purity (see Materials and Methods). The purified proteins run at their corresponding molecular weight in reducing SDS-PAGE. (B-C) CD melt spectroscopy shows that both H1N1 HA1 (1–330) (B) and H1N1 HA (1–480) (C) are properly folded. Both H1N1 HA proteins, at a concentration of 0.5 mg/ml in 20 mM PBS, pH 7.2, were subjected to heating at 0.5°C/min increments. The protein unfolding kinetics was measured at 222 nm using a J-715 Circular Dichroism system (JASCO corp., Easton, MD). (D-E) Superdex S-200 gel filtration chromatography of purified H1N1 HA proteins from E.coli. The panels present superimposed elution profiles of purified HA proteins (red line) overlaid with calibration standards (grey line). (D) The H1N1 HA (1–330) protein purified from bacterial cells existed as approximately 20% high-molecular-mass oligomer (>600 kDa), 45% trimer (∼110 kDa) and 35% monomer (34kDa) (red line). (E) H1N1 HA (1–480) is present only as a monomer (50kDa). (F-G) Binding kinetics of purified H1N1 HA proteins in a SPR based receptor binding assay. Steady-state equilibrium analysis of different H1N1-HA proteins to fetuin and its asialylated counterpart (Asialo-fetuin) was analyzed at 25°C using a ProteOn surface plasmon resonance biosensor (BioRad Labs). Samples of purified H1N1-HA proteins (10 µg/ml) were injected simultaneously over a mock surface to which no protein was bound, followed by the Asialofetuin in (F) or Fetuin in (G) immobilized on a sensor chip through the free amine group, and onto a blank flow cell, free of protein. Binding kinetics and data analysis were performed using ProteOn system surface plasmon resonance biosensor instrument (BioRad Labs, Hercules, CA). (H) Agglutination of human RBCs by properly folded bacterial H1N1 HA (1–330) protein. Serial dilutions of purified HA proteins or virus were mixed with washed RBC and incubated to analyze the receptor binding and cross-linking of human RBC. Virus H1N1xPR8 A/California/07/2009 (X-179A) was used as a control. Strong hemagglutination was observed for bacterial H1N1 HA (1–330) but not with either bacterial H1N1 HA (1–480) or mammalian H1N1 HA0.

Surender Khurana, et al. PLoS One. 2010;5(7):e11548.

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