Volume 15, Issue 2, Pages 411-421 (February 2007) Enhanced Cellular Immune Responses Elicited by an Engineered HIV-1 Subtype B Consensus-based Envelope DNA Vaccine  Jian Yan, Hanna Yoon, Sanjeev Kumar, Mathura P Ramanathan, Natasha Corbitt, Michele Kutzler, Anlan Dai, Jean D Boyer, David B Weiner  Molecular Therapy  Volume 15, Issue 2, Pages 411-421 (February 2007) DOI: 10.1038/sj.mt.6300036 Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 1 Design, construction, and expression of a subtype B consensus-based envelope gene. (a) Comparison of the aa sequences of EY2E1-B and EK2P-B. The IgE leader sequence is underlined. The boxed regions show variable regions. *Six important residues involved in CCR5 utilization. An arrow indicated the cleavage site. The transmembrane domain is shown by the dotted line. (b) Western blotting analysis of EY2E1-B and EK2P-B genes. RD cells were transfected with different plasmids. After 48 h, cell lysates were collected. Samples were analyzed by Western blotting and probed with HIV-1 gp120 monoclonal (2G12). As for loading control, the blot was stripped and reprobed with a monoclonal anti-actin antibody (c) Immunofluorescence assay of EY2E1-B and EK2P-B genes. The transfected RD cells expressing envelope proteins showed typical red fluorescence. HIV-1 envelope-specific monoclonal antibody F105 served as the source of primary antibody. Molecular Therapy 2007 15, 411-421DOI: (10.1038/sj.mt.6300036) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 2 Total IgG antibody titers in the sera of the immunized BALB/c mice. (a) The measurement of subtype B envelope-specific antibody responses. The significant P-value from the one-tailed t-test for the pEY2E1-B-immunized group versus the pEK2P-B-immunized group is 0.023. (b) The measurement of subtype A/E envelope-specific antibody responses. (c) The measurement of subtype C envelope-specific antibody responses. Humoral immune responses after immunization with pEY2E1-B and pEK2P-B were detected by enzyme-linked immunosorbent assay. Mice from each group (n=5) were bled 1 week after the third immunization, and equally pooled sera were diluted and analyzed as described in Materials and Methods. Pooled sera collected from mice immunized with pVAX were used as a control. Absorbance (OD) was measured at 450 nm. Each data point represents averaged five OD values from five mice sera per group and values represent the mean of enzyme-linked immunosorbent assay obtained in three separate studies. Molecular Therapy 2007 15, 411-421DOI: (10.1038/sj.mt.6300036) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 3 Induction of cell-mediated immune responses by pEY2E1-B in both BALB/c and HLA-A2 Tg mice. Frequencies of subtype B consensus envelope-specific IFN-γ SFUs/million splenocytes after DNA vaccination with pEY2E1-B and pEK2P-B were determined by ELISpot assay in both (a) BALB/c mice and (c) Tg mice. Frequencies of CD8 depleted, subtype B consensus envelope-specific IFN-γ SFUs/million splenocytes after DNA vaccination with pEY2E1-B and pEK2P-B were also determined in both (b) BALB/c and (d) Tg mice. The slenocytes were isolated from individual immunized mice (five mice per group) and stimulated in vitro with overlapping consensus subtype B envelope peptide pools. Backbone pVAX-immunized mice were included as a negative control. The values are the means±SDs of the means of IFN-γ SFUs. (e) Characterization of subtype B consensus envelope-specific dominant epitopes. The splenocytes collected from pEY2E1-B- and pEK2P-B-vaccinated BALB/c mice, respectively, were cultured with 29 HIV-1 subtype B consensus envelope peptide pools for 24 h. IFN-γ-secreting cells were determined by ELISpot assay as described above. Molecular Therapy 2007 15, 411-421DOI: (10.1038/sj.mt.6300036) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 4 Cross-reactivity induced by pEY2E1-B in BALB/c mice. (a) The additive T-cell immune responses in BALB/c mice induced by vaccination with pEY2E1-B and pEK2P-B against four individual peptide pools of HIV-1 MN envelope peptides, (b) HIV-1 group M peptides, (c) subtype C consensus envelope peptides, and (d, e) two subtype C isolate envelope peptides were measured by IFN-γ ELISpot assay. Backbone pVAX-immunized mice were included as a negative control. Molecular Therapy 2007 15, 411-421DOI: (10.1038/sj.mt.6300036) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 5 Cross-reactivity induced by pEY2E1-B in C57BL/6 mice. (a) The additive T-cell immune responses in C57BL/6 mice induced by vaccination with pEY2E1-B and pEK2P-B against four individual peptide pools of HIV-1 MN envelope peptides, (b) HIV-1 group M peptides, (c) subtype C consensus envelope peptides, and (d, e) two subtype C isolate envelope peptides were also measured. Backbone pVAX-immunized mice were included as a negative control. Molecular Therapy 2007 15, 411-421DOI: (10.1038/sj.mt.6300036) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 6 Cross-reactivity induced by pEY2E1-B in HLA-A2 Tg mice. (a) The additive T-cell immune responses in HLA-A2 Tg mice induced by vaccination with pEY2E1-B and pEK2P-B against four individual peptide pools of HIV-1 MN envelope peptides, (b) HIV-1 group M peptides, (c) subtype C consensus envelope peptides, and (d, e) two subtype C isolate envelope peptides were also measured. Backbone pVAX-immunized mice were included as a negative control. Molecular Therapy 2007 15, 411-421DOI: (10.1038/sj.mt.6300036) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions

Figure 7 Enhanced breadth of cross-reactivity induced by pEY2E1-B in BALB/c and HLA-A2 Tg mice. Characterization of subtype B MN envelope-specific dominant epitopes in both (a) BALB/c and (b) HLA-A2 Tg mice immunized with EY2E1-B and EK2P-B immunogens. The splenocytes collected from pEY2E1-B- and pEK2P-B-vaccinated BALB/c and Tg were cultured with 29 HIV-1 subtype B MN envelope peptide pools for 24 h. IFN-γ-secreting cells were determined by ELISpot assay as described above. Molecular Therapy 2007 15, 411-421DOI: (10.1038/sj.mt.6300036) Copyright © 2007 The American Society of Gene Therapy Terms and Conditions