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1.
FIG. 4.

FIG. 4. From: Human Immunodeficiency Virus Type 1 Env Trimer Immunization of Macaques and Impact of Priming with Viral Vector or Stabilized Core Protein .

V3 peptide competition mapping of neutralizing activity. V3 peptide mapping was performed on individual serum samples against MN, HXBc2, and SF162. The V3 peptide sequence used to map neutralizing activity targeting the V3 loop was based on the YU2 sequence (TRPNNNTRKSINIGPGRALYTTG) (filled circles). A scrambled V3 peptide (IGPGRATRPNNNFYTTGTRKSIH) was used as a negative control (open circles). The results from these experiments are shown as percent inhibition of the ID50 neutralization values where only values over 50% are considered a reliable signal. Values in excess of a 50% reduction of ID50 neutralization are indicated by the dotted line.

Andreas Mörner, et al. J Virol. 2009 January;83(2):540-551.
2.
FIG. 2.

FIG. 2. From: Human Immunodeficiency Virus Type 1 Env Trimer Immunization of Macaques and Impact of Priming with Viral Vector or Stabilized Core Protein .

Env binding antibodies in serum from immunized animals. The titers of Env-binding antibodies were measured in sera 2 weeks after each immunization using a standard ELISA with gp120-coated wells. The OD50 titer for each sample was calculated by interpolating from the mean OD50 value calculated from controls: [(ODmax − ODmin)/2] + ODmin. The results for individual animals, as well as group means, are shown. Preimmune Env-directed reactivity in sera diluted 100-fold was also determined and was shown not to differ between the groups (see Fig. S1A in the supplemental material). “T” indicates gp140-F trimer protein immunization, “S” indicates SFV-gp140-F immunization, and “C” indicates stable core protein immunization.

Andreas Mörner, et al. J Virol. 2009 January;83(2):540-551.
3.
FIG. 5.

FIG. 5. From: Human Immunodeficiency Virus Type 1 Env Trimer Immunization of Macaques and Impact of Priming with Viral Vector or Stabilized Core Protein .

ELISA using different Env proteins for coating. (A) Serum samples from 2 weeks after the fifth immunization were tested in an ELISA using either native or denatured gp120 for coating. The native versus denatured OD50 titer ratio for each individual animal is shown. (B) Binding antibodies against the stable core protein (upper panel) or gp140-F (lower panel) used for coating in the ELISA plates were measured in serum samples from individual animals 2 weeks after the second and fourth immunizations and plotted as group means. “T” refers to gp140-F trimer protein immunization, “S” refers to SFV-gp140-F immunization, and “C” refers to stable core protein immunization.

Andreas Mörner, et al. J Virol. 2009 January;83(2):540-551.
4.
FIG. 3.

FIG. 3. From: Human Immunodeficiency Virus Type 1 Env Trimer Immunization of Macaques and Impact of Priming with Viral Vector or Stabilized Core Protein .

Neutralizing antibody responses against clade B viruses. Serum samples from each animal taken 2 weeks after the second, fourth, and fifth immunizations were analyzed for neutralizing activity using the TZM-bl/Luc neutralization assay and the following Env glycoproteins: MN, HXBc2, SF162, BaL.01, SS1196.01, JRFL, and YU2 (clade B). Serial serum dilutions from individual monkeys were tested, and the data are presented as 50% neutralization titers (ID50). Neutralizing ID50 titers above 20 are considered positive, as indicated by the dotted lines. “T” refers to gp140-F trimer protein immunization, “S” refers to SFV-gp140-F immunization, and “C” refers to stable core protein immunization.

Andreas Mörner, et al. J Virol. 2009 January;83(2):540-551.
5.
FIG. 6.

FIG. 6. From: Human Immunodeficiency Virus Type 1 Env Trimer Immunization of Macaques and Impact of Priming with Viral Vector or Stabilized Core Protein .

Vaccine-induced T-cell responses. HIV-1 Env-directed T-cell responses were measured by IFN-γ ELISPOT after restimulation of PBMC with overlapping peptides. Two different peptide pools based on the YU2 gp140 sequence were used: “gp140-F core” covered the conserved regions of gp120, corresponding to the stable core immunogen, and “gp140-F non-core” covered the remaining parts of YU2 gp140. (A) gp140-F core (light purple) and noncore (dark purple) responses before immunization (Pre) and 2 weeks after the second (2×T) and fourth (4×T) immunizations are shown. “T” refers to gp140-F trimer protein immunization, “S” refers to SFV-gp140-F immunization, and “C” refers to stable core protein immunization. (B) The percent core-specific response of the total gp140-F response after the second and fourth immunizations is shown. (C) PBMC collected 2 weeks after immunization 4 were depleted of CD8+ T cells by magnetic cell sorting. Total PBMC (PBMC) and CD8+ T-cell-depleted PBMC (−CD8) were restimulated with the gp140-F core and gp140-F noncore peptide pools. Shown are the cumulative gp140-F core and noncore responses. The viability in both total PBMC and CD8+ T-cell-depleted PBMC was >93%. The CD8+ T-cell-depleted PBMC contained <0.2% CD8+ T cells, as measured by flow cytometry.

Andreas Mörner, et al. J Virol. 2009 January;83(2):540-551.
6.
FIG. 1.

FIG. 1. From: Human Immunodeficiency Virus Type 1 Env Trimer Immunization of Macaques and Impact of Priming with Viral Vector or Stabilized Core Protein .

Design and biochemical characterization of HIV-1 envelope glycoproteins. (A) Linear (sequence) and tertiary (structure) schematic representation of antigens used for immunization. Trimers of gp140-F were based on YU2 and include the full-length gp120 sequence with N and C termini (N and C), variable regions (V1/V2/V3/V4 and V5), and the gp41 ectodomain (gray). They are cleavage defective (the REKR sequence at the cleavage site was changed to SEKS to maintain a covalent gp120-gp41 association) and contain a heterologous foldon trimerization motif (F) (yellow). Stable cores were based on HXBc2 and lack N and C termini, the V1/V2/V3 regions, and gp41. They were modified by structure-based design to contain pocket-filling mutations (M95W, T257S, A433M, and S375W, which eliminates F105 recognition) (13, 56) and extra cysteine bonds between amino acids 96 and 275 and amino acids 109 and 428, as indicated by dotted lines in the linear schematic and green bars in the tertiary cartoon; these disulfides stabilize the CD4-bound conformation of gp120 (56). The inner domain of the HXBc2 core is colored blue and the outer domain red. Numbering of amino acids is based on the HXBc2 numbering convention. (B) Purified proteins were analyzed for their antigenic profiles by immunoprecipitation with the three monoclonal antibodies: 17b (coreceptor site directed), F105 (CD4bs directed), and 447-52D (V3 directed). The eluted proteins were resolved on a NuPAGE 4-to-12% bis-Tris gel (left panel). (C) The oligomeric status of the proteins was analyzed by blue native gel electrophoresis. Thyroglobulin and ferritin (GE Healthcare) were used as molecular mass markers.

Andreas Mörner, et al. J Virol. 2009 January;83(2):540-551.

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