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. 2010 Sep 1:7:71.
doi: 10.1186/1742-4690-7-71.

Broader HIV-1 neutralizing antibody responses induced by envelope glycoprotein mutants based on the EIAV attenuated vaccine

Lianxing Liu  1 , Yanmin Wan, Lan Wu, Jianping Sun, Huiguang Li, Haishan Li, Liying Ma, Yiming Shao
Affiliations

Broader HIV-1 neutralizing antibody responses induced by envelope glycoprotein mutants based on the EIAV attenuated vaccine

Lianxing Liu et al. Retrovirology. .

Abstract

Background: In order to induce a potent and cross-reactive neutralizing antibody (nAb), an effective envelope immunogen is crucial for many viral vaccines, including the vaccine for the human immunodeficiency virus (HIV). The Chinese equine infectious anemia virus (EIAV) attenuated vaccine has controlled the epidemic of this virus after its vaccination in over 70 million equine animals during the last 3 decades in China. Data from our past studies demonstrate that the Env protein of this vaccine plays a pivotal role in protecting horses from both homologous and heterogeneous EIAV challenges. Therefore, the amino acid sequence information from the Chinese EIAV attenuated vaccine, in comparison with the parental wild-type EIAV strains, was applied to modify the corresponding region of the envelope glycoprotein of HIV-1 CN54. The direction of the mutations was made towards the amino acids conserved in the two EIAV vaccine strains, distinguishing them from the two wild-type strains. The purpose of the modification was to enhance the immunogenicity of the HIV Env.

Results: The induced nAb by the modified HIV Env neutralized HIV-1 B and B'/C viruses at the highest titer of 1:270. Further studies showed that a single amino acid change in the C1 region accounts for the substantial enhancement in induction of anti-HIV-1 neutralizing antibodies.

Conclusions: This study shows that an HIV envelope modified by the information of another lentivirus vaccine induces effective broadly neutralizing antibodies. A single amino acid mutation was found to increase the immunogenicity of the HIV Env.

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Figures

Figure 1
Figure 1
Consensus mutations and schematic structures are similar between EIAV and HIV-1. a) Sequence analysis show 10 consensus amino acid mutational sites that have been identified between two Chinese vaccine-derived wild-type EIAV strains and two vaccine virus strains in the EIAV Env region ("--" means that this amino acid was deleted). b) Schematic illustration of gp145 mutants. The figure after the M represents the region of mutations made in the CN54 gp145. c) Schematic figure of the EIAV D510 V3, V4 regions and the HIV-1 CN54 V1, V2 regions. The left figure shows the EIAV V3, V4 regions; the right figure shows the HIV-1 V1, V2 regions. N-Glycosylation sites are shown as branched lines.
Figure 2
Figure 2
Specific binding antibody titer. a) Vaccine inoculation schedule of mice. All groups were inoculated with DNA vaccine at Weeks 0, 2, 4 and 6 and then sacrificed at Week 9 to assess cellular and humoral immune responses. b, c & d) The specific binding antibody titer induced by DNA vaccines. Antibody reactivity was then determined by measuring the optical density (OD) at 492 nm, and endpoint titers were determined by the last dilution whose OD was >two-times than that at the average corresponding dilution of mice sera immunized with SV1.0. The Y value is the log value of the endpoint titers. The significance of differences among the different groups was calculated using a statistical method of one-way analysis of variance (GraphPad prism4.0); * p < 0.05, ** p < 0.01. We obtained data from three experiments using fresh sera.
Figure 3
Figure 3
Inhibition of HIV by sera of immunized guinea pigs at the titer of 1:10. a) Vaccine inoculation schedule of guinea pigs. All groups were inoculated with DNA vaccine at weeks 0, 2, 4 and then boosted with rTV at week 10. Sera after last immunization were collected at week 14 and 16. b) Comparative inhibition of HIV-1 infection by sera collected at week 16 from mock-, gp145- and gp145-10M-immunized guinea pigs. The neutralizing experiment was conducted by using a panel of clinical HIV-1 isolates from PBMCs in TZM-bl cells. The dotted line in the figure indicates the 50% inhibition rate.
Figure 4
Figure 4
Neutralizing antibody titers against HIV-1 of immunized guinea pigs' sera. End-point neutralizing antibody titers of HIV-1 by sera from mock-, gp145- and gp145-10M-immunized guinea pigs. Sera were collected at six weeks after the last immunization for testing. The neutralizing experiment was conducted by using a panel of clinical isolates in PBMC cells (a) and a panel of tier 2-3 peudeovirus in TZM-b1 cells (b). The 50% inhibitory dose (ID50) was defined as the plasma dilution.
Figure 5
Figure 5
Linear antibody epitope mapping. PLL-ELISA was used to detect the responses of the antibody against each linear peptide. The concentration of each peptide of HIVCN54gp145 was 10 μg/ml. In these epitopes, seven were defined as positive (> two-fold background). Epitopes p4838, p4859 and p4860 were only identified in the SV145 group. P4876 was only defined in the SV145-10 M group. The grey line in the figure indicates the threshold value (0.22), which is two times than the average OD values (0.11) from mock controls. Five mutation regions were labeled.
Figure 6
Figure 6
Specific IFN-gamma secretion detected by ELISPOT. The T cell immunity was quantified with an IFN-γ-based ELISPOT assay with SHIVchn19 (20-mer) peptides ENV1 (4830-4871) or ENV2 pools (4872-4913). a, e & i) Elispot results in different immunized groups stimulated with stimuli as indicated on the left. b, f & k) Number of SFC in one million splenocytes stimulated by the ENV1 pool. c, g & l) Number of SFC in one million splenocytes stimulated by the ENV2 pool. d, h & m) Env-specific T cell responses against these two peptide pools were compiled together as the total T cell responses for each mouse and were graphed into groups. The mock group generated <50 SFCs/106 splenocytes. The significance of differences among the different groups was calculated using a statistical method of one-way analysis of variance (GraphPad prism4.0); SFC, spot forming cell; * p < 0.05, ** p < 0.01. The error bars indicate SEM (the standard error of the mean).
Figure 7
Figure 7
The secondary structure prediction and hydrophilicity changes of antigens. AnthePro5.0 software was applied to predict the secondary structure and hydrophilicity changes of gp145 and gp145M1. The red rectangle labels the changed profiles. a & b) The secondary structure predictions of gp145 and gp145M1. α-helix in blue bar; β-sheet in orange bar; β-turn in green bar; others in black bar. c & d) The difference in hydrophilicity between gp145 and gp145M1. The Y-axis shows the value of predicted hydrophilicity.
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