Article

Analysis of the human immunodeficiency virus type 1 gp41 membrane proximal external region arrayed on hepatitis B surface antigen particles.

Structural Virology Section, Vaccine Research Center, NIAID, NIH, Bethesda, MD 20892-3005, USA.
Virology (Impact Factor: 3.28). 04/2008; 373(1):72-84. DOI: 10.1016/j.virol.2007.11.005
Source: PubMed

ABSTRACT Vaccine immunogens derived from the envelope glycoproteins of the human immunodeficiency virus type 1 (HIV-1) that elicit broad neutralizing antibodies remain an elusive goal. The highly conserved 30 amino-acid membrane proximal external region (MPER) of HIV gp41 contains the hydrophobic epitopes for two rare HIV-1 broad cross-reactive neutralizing antibodies, 2F5 and 4E10. Both these antibodies possess relatively hydrophobic HCDR3 loops and demonstrate enhanced binding to their epitopes in the context of the native gp160 precursor envelope glycoprotein by the intimate juxtaposition of a lipid membrane. The hepatitis B surface antigen (HBsAg) S1 protein forms nanoparticles that can be utilized both as an immunogenic array of the MPER and to provide the lipid environment needed for enhanced 2F5 and 4E10 binding. We show that recombinant HBsAg particles with MPER (HBsAg-MPER) appended at the C-terminus of the S1 protein are recognized by 2F5 and 4E10 with high affinity compared to positioning the MPER at the N-terminus or the extracellular loop (ECL) of S1. Addition of C-terminal hydrophobic residues derived from the HIV-1 Env transmembrane region further enhances recognition of the MPER by both 2F5 and 4E10. Delipidation of the HBsAg-MPER particles decreases 2F5 and 4E10 binding and subsequent reconstitution with synthetic lipids restores optimal binding. Inoculation of the particles into small animals raised cross-reactive antibodies that recognize both the MPER and HIV-1 gp160 envelope glycoproteins expressed on the cell surface; however, no neutralizing activity could be detected. Prime:Boost immunization of the HBsAg-MPER particles in sequence with HIV envelope glycoprotein proteoliposomes (Env-PLs) did not raise neutralizing antibodies that could be mapped to the MPER region. However, the Env-PLs did raise anti-Env antibodies that had the ability to neutralize selected HIV-1 isolates. The first generation HBsAg-MPER particles represent a unique means to present HIV-1 envelope glycoprotein neutralizing determinants to the immune system.

0 Bookmarks
 · 
104 Views
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: The structure of the HIV-1 Envelope membrane-proximal external region (MPER) is influenced by its association with the lipid bilayer on the surface of virus particles and infected cells. To develop a replicating vaccine vector displaying MPER sequences in association with membrane, Env epitopes recognized by the broadly neutralizing antibodies 2F5, 4E10, or both were grafted into the membrane-proximal stem region of the vesicular stomatitis virus (VSV) glycoprotein (G). VSV encoding functional G-MPER chimeras based on G from the Indiana or New Jersey serotype propagated efficiently although grafting of both epitopes (G-2F5-4E10) modestly reduced replication and resulted in the acquisition of one to two adaptive mutations in the grafted MPER sequence. Monoclonal antibodies 2F5 and 4E10 efficiently neutralized VSV G-MPER vectors and bound to virus particles in solution, indicating that the epitopes were accessible in the pre-attachment form of the G-MPER chimeras. Overall, our results showed that the HIV Env MPER could functionally substitute for the VSV G stem region implying that both perform similar functions even though they are from unrelated viruses. Furthermore, we found that the MPER sequence grafts induced low but detectable MPER-specific antibody responses in rabbits vaccinated with live VSV, although additional vector and immunogen modifications or use of a heterologous prime-boost vaccination regimen will be required to increase the magnitude of the immune response.
    AIDS research and human retroviruses 03/2014; 30(11). DOI:10.1089/AID.2013.0206 · 2.18 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Developing a vaccine that overcomes the diversity of HIV-1 is likely to require a strategy that directs antibody (Ab) responses toward conserved regions of the viral Envelope (Env). However, the generation of neutralizing Abs (NAbs) targeting these regions through vaccination has proven to be difficult. One conserved region of particular interest is the membrane proximal external region (MPER) of Env located within the gp41 ectodomain. In order to direct the immune response to this region, the MPER and gp41 ectodomain were expressed separately as N-terminal fusions to the E2 protein of Geobacillus stearothermophilus. The E2 protein acts as a scaffold by self-assembling into 60-mer particles, displaying up to 60 copies of the fused target on the surface. Rabbits were immunized with E2 particles displaying MPER and/or the gp41 ectodomain in conjunction with DNA encoding full-length gp160. Only vaccines including E2 particles displaying MPER elicited MPER-specific Ab responses. NAbs were elicited after two immunizations that largely targeted the V3 loop. To overcome V3 immunodominance in the DNA component, E2 particles displaying MPER were used in conjunction with gp160 DNA lacking hypervariable regions V2, V3, or combined V1V2V3. All rabbits had HIV binding Ab responses and NAbs following the second vaccination. Using HIV-2/HIV-1 MPER chimeric viruses as targets, NAbs were detected in 12/16 rabbits after three immunizations. Low levels of NAbs specific for Tier 1 and 2 viruses were observed in all groups. This study provides evidence that co-immunizing E2 particles displaying MPER and gp160 DNA can focus Ab responses toward conserved regions of Env.
    PLoS ONE 12/2014; 9(12):e113463. DOI:10.1371/journal.pone.0113463 · 3.53 Impact Factor
  • Source
    [Show abstract] [Hide abstract]
    ABSTRACT: Continued efforts to define the immunogenic properties of the HIV-1 envelope glycoproteins (Env) are needed to elicit effective antibody (Ab) responses by vaccination. HIV-1 is a highly neutralization-resistant virus due to conformational and glycan shielding of conserved Ab determinants on the virus spike. Elicitation of broadly neutralizing Abs that bind poorly accessible epitope regions on Env is therefore extremely challenging and will likely require selective targeting of specific sub-determinants. To evaluate such approaches there is a pressing need for in vivo studies in both large and small animals, including mice. Currently, most mouse immunization studies are performed in the BALB/c strain; however, the C57BL/6 strain offers improved possibilities for mechanistic studies due to the availability of numerous knock-out strains on this genetic background. Here, we compared Env immunogenicity in BALB/c and C57BL/6 mice and found that the magnitude of the antigen-specific response was somewhat lower in C57BL/6 than in BALB/c mice by ELISA but not significantly different by B cell ELISpot measurements. We then established protocols for the isolation of single Env-specific memory B cells and germinal center (GC) B cells from immunized C57BL/6 mice to facilitate future studies of the elicited response at the monoclonal Ab level. We propose that these protocols can be used to gain an improved understanding of the early recruitment of Env-specific B cells to the GC as well as the archiving of such responses in the memory B cell pool following immunization.
    Viruses 09/2014; 6(9):3400-3414. DOI:10.3390/v6093400 · 3.28 Impact Factor

Full-text (2 Sources)

Download
26 Downloads
Available from
May 30, 2014