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Haynes Progress Report Abstracts continued

Submitted February 11, 2011 (Interim Report)

Our overall goal is to acquire proof of concept data that manipulation of the immunoregulatory controls of B cell immune responses to HIV-1 Env, coupled with enhanced immunogen design, can lead to safe induction of broadly reactive neutralizing antibody responses. The CAVD team has been successful and demonstrated that central and peripheral tolerance mechanisms control the expression of gp41 membrane proximal external region (MPER) neutralizing antibodies in knock-in mice, that non-neutralizing MPER antibodies bind to the post-fusion six helix bundle of gp41, and that germline antibodies bind to a host protein in both mouse and human with sequences homologous to the HIV gp41 MPER. From this work, it appears that what is required is directed affinity maturation of a gp41 neutralizing antibody response, as well as breaking peripheral anergy of otherwise unresponsive B cells. MPER peptides in membrane liposomes have been designed to mimic virions, and potent adjuvants have been incorporated into MPER-liposomes. Thus, one lead candidate as an MPER immunogen is liposomes with viral lipids (“virosomes”) containing the gp41 intermediate form of the MPER that may be primed with an endogenous MPER homologous host antigen. Currently, the work is focused on immunization studies in rhesus macaques to focus the antibody response on the MPER neutralizing epitopes, and on MPER second generation immunogen design. For gp120 epitopes, this CAVD has identified 3 transmitted founder Envs as candidate vaccines that are both superior as antigenic and immunogenic Envs compared to 28 others tested. Finally, the CAVD team is working on defining the correlates of protective immunity to HIV in the RV144 trial samples.

 
Submitted September 15, 2010

Our overall goal is to acquire proof of concept data that manipulation of the immunoregulatory controls of B cell immune responses to HIV-1 Env, coupled with enhanced immunogen design, can lead to safe induction of broadly reactive neutralizing antibody responses. The CAVD team has been successful and demonstrated that central and peripheral tolerance mechanisms control the expression of gp41 membrane proximal external region (MPER) neutralizing antibodies, that non-neutralizing MPER antibodies bind to the post-fusion six helix bundle of gp41, and that germline antibodies bind to a host protein in both mouse and human with sequences homologous to the HIV gp41 MPER. From this work, it appears that what is required is directed affinity maturation of a gp41 neutralizing antibody response, as well as breaking peripheral anergy of otherwise unresponsive B cells. MPER peptides in membrane liposomes have been designed to mimic virions, and potent adjuvants have been incorporated into MPER-liposomes. Thus, one lead candidate as an MPER immunogen is liposomes with viral lipids (“virosomes”) containing the gp41 intermediate form of the MPER that may be primed with an endogenous MPER homologous host antigen.  Currently the work is focused on adding TLR ligands and other immunostimulatory molecules to the liposomes in order to target the B cells capable of making broadly neutralizing antibodies to the gp41 MPER. For gp120 epitopes, this CAVD has identified 3 transmitted founder Envs as candidate vaccines that are both superior as antigenic and immunogenic Envs compared to 28 others tested.

 
Submitted February 1, 2010 (Interim Report)

Our overall goal is to acquire proof of concept data that manipulation of the immunoregulatory controls of B cell immune responses to HIV-1 Env, coupled with enhanced immunogen design, can lead to safe induction of broadly reactive neutralizing antibody responses. We are using a two armed approach to the problem of induction of antibodies that broadly neutralize HIV.

The Kelsoe, Haynes and Thorpe laboratories are developing immunogen formulations that trigger B cells normally tolerant to the desired envelope epitopes and regions. Garnett Kelsoe is determining the origins, development, physiology, and fates of marginal zone, transitional and B1 B cell populations in animal models including mice and non-human primates. Laurent Verkoczy has constructed broadly neutralizing 2F5 and 4E10 human mAb VH +VL knock-in mice.  Importantly the mouse models developed by Kelsoe as well as by Verkoczy are now being used for immunization with the group’s lead immunogens to determine the immunoregulatory mechanisms involved in control of these types of antibodies.  Haynes is determining the role of tolerance mechanisms, and TLR signaling on control of broadly-reactive B cell activation, and Philip Thorpe is determining the role of lipid binding of anti-HIV antibodies and anti-phosphatidylserine (PS) autoantibodies to protection from HIV infection. 

The Harrison, Alam, Spicer, Shaw, Robinson and Hahn laboratories are developing immunogens that are in native conformations and are being tested for induction of neutralizing antibodies.  These include HIV Env immunogens with a spectrum of affinities for binding to broadly reactive neutralizing antibodies, and immunogens with low entropic barriers to Mab binding and therefore are thermodynamically stable. Steve Harrison, Bing Chen and Larry Liao have made single chain Fv 4E10 antibodies and whole IgG1 2F5 Mabs with mutations that selectively eliminate gp41 or lipid reactivity.  They have demonstrated that both 2F5 and 4E10 require the capacity to bind to lipids to neutralize HIV-1. They are now using the Fvs and Mabs in co-crystallization efforts with trimeric gp120/gp41 constructs.  Munir Alam has designed lead candidate gp41 peptide lipid conjugates using 4E10 and 2F5 epitopes peptides, and as well has the lead candidate immunogen liposomes in which the gp41 intermediate form of Chen and Harrison have been incorporated into liposomes with TLR ligands and cytokines.  Spicer is studying the lipid-peptide conjugates for their structures by NMR. George Shaw and Beatrice Hahn have constructed HIV-1/HIV-2 chimeras with HIV-2 neutralizing determinants in HIV-1 scaffolds, to study the neutralization of HIV-2 with James Robinson. Dimiter Dimitrov at the NCI has used phage-displayed library technology to isolate a 2F5-like monoclonal antibody from a patient with 2F5-like broadly neutralizing antibodies

 

Submitted September 15, 2009

Our overall goal is to acquire proof of concept data that manipulation of the immunoregulatory controls of B cell immune responses to HIV-1 Env, coupled with enhanced immunogen design, can lead to safe induction of broadly reactive neutralizing antibody responses. We are using a two armed approach to the problem of induction of antibodies that broadly neutralize HIV.

The Kelsoe, Haynes and Thorpe laboratories are developing immunogen formulations that trigger B cells normally tolerant to the desired envelope epitopes and regions. Garnett Kelsoe is determining the origins, development, physiology, and fates of marginal zone, transitional and B1 B cell populations in animal models including mice and non-human primates. Laurent Verkoczy has constructed broadly neutralizing 2F5 and 4E10 human mAb VH +VL knock-in mice. Importantly the mouse models developed by Kelsoe as well as by Verkoczy are now being used for immunization with the group’s lead immunogens to determine the immunoregulatory mechanisms involved in control of these types of antibodies. Haynes is determining the role of tolerance mechanisms, and TLR signaling on control of broadly-]reactive B cell activation, and Philip Thorpe is determining the role of lipid binding of anti-HIV antibodies and anti-phosphatidylserine (PS) autoantibodies to protection from HIV infection. The team has recently found that non-pathogenic anti-lipid antibodies that do not require beta-2-glycoprotein-1 for lipid binding do prevent HIV-1 and SHIVSF162P3 (and all R5 HIVs tested from infecting PBMC in vitro, and a prototype of non-pathogenic anti-lipid antibodies will be studied in non-human primates in vivo for the ability to protect against R5 SHIV infection.

The Harrison, Alam, Spicer, Shaw, Robinson and Hahn laboratories are developing immunogens that are in native conformations and are being tested for induction of neutralizing antibodies. These include HIV Env immunogens with a spectrum of affinities for binding to broadly reactive neutralizing antibodies, and immunogens with low entropic barriers to Mab binding and therefore are thermodynamically stable. Steve Harrison, Bing Chen and Larry Liao have made single chain Fv 4E10 antibodies and whole IgG1 2F5 Mabs with mutations that selectively eliminate gp41 or lipid reactivity. They have demonstrated that both 2F5 and 4E10 require the capacity to bind to lipids to neutralize HIV-1. They are now using the Fvs and Mabs in co-crystallization efforts with trimeric gp120/gp41 constructs. Munir Alam has designed lead candidate gp41 peptide lipid conjugates using 4E10 and 2F5 epitopes peptides, and as well has as the lead candidate immunogen liposomes in which the gp41 intermediate form of Chen and Harrison have been incorporated into liposomes with TLR ligands and cytokines. Spicer is studying the lipid-peptide conjugates for their structures by NMR. George Shaw and Beatrice Hahn have constructed HIV-1/HIV-2 chimeras with HIV-2 neutralizing determinants in HIV-1 scaffolds, to study the neutralization of HIV-2 with James Robinson. The Team led by Victor Chalwe at the Tropical Disease Research Laboratory in Ndola, Zambia has completed on schedule providing the CAVD with PBMC and plasma on 135 chronically-infected subjects for mapping epitopes of broadly neutralizing antibodies for immunogen design. Dimiter Dimitrov at the NCI has used phage-displayed library technology to isolate a 2F5-like monoclonal antibody from a patient with 2F5-like broadly neutralizing antibodies. Finally, we have established a collaboration with Andrew Fire and Scott Boyd at Stanford to probe the VH repertoire.

 
Submitted February 1, 2009 (Interim Report)

Our goal is to acquire proof of concept data that manipulation of the immunoregulatory controls of B cell immune responses to HIV-1 Env, coupled with enhanced immunogen design, can lead to safe induction of broadly reactive neutralizing antibody responses. This year has been a pivotal year for the Haynes VDC, in that we have achieved the first level of proof of concept for the hypothesis that tolerance mechanisms are indeed regulating antibody responses to the gp41 membrane proximal external region (MPER), and our structural work has produced a lead candidate gp41 MPER immunogen. 

The evidence for tolerance control of MPER antibodies comes from two projects.  Garnett Kelsoe has developed a mouse model, the culture derived (CD) B cell model , wherein C57BL/6 mouse bone marrow B cells are cultured in the absence of bone marrow tolerance mechanisms, and allowed to differentiate to immature B cells.  Transfer of these B cells into immunodeficient mice allows the mice to be reconsitituted with this non-tolerized B cell population.  Kelsoe has demonstrated that  B cells in wildtype C57BL/6 mice, when immunized with HIV envelope gp41 MPER, do not make MPER antibodies, whereas culture derived C57BL/6 B cells transplanted into  immunodeficient mice, when similarly immunized, do make MPER antibodies.  Kelsoe now has over 250 MPER-specific murine monoclonal antibodies from these mice to determine lipid reactivity and HIV neutralizing activity. 

The second project with evidence of tolerance control of MPER antibodies comes from Laurent Verkoczy with mice “knocked-in” with the VH and VL chain genes of the broadly neutralizing MPER antibodies 2F5 and 4E10. Thus far, the characterization of the 2F5 HC (heavy chain) knock-in mice is complete and shows that the mouse handles the 2F5 HC as if it were an autoantibody and is regulated by tolerance mechanisms. Evaluation of the 4E10 HC knock-in mouse is underway, as is breeding of both the 4E10 and 2F5 HC mice with their respective LC (light chain) transgenic counterparts to create mice expressing full 2F5 or 4E10 H+L mice.  Evaluation of these animals should provide the required insight into how to best induce these types of anti-HIV envelope antibodies.

The Harrison, Alam, Spicer, Shaw, Robinson and Hahn laboratories are developing immunogens that are in native conformations and are being tested for induction of neutralizing antibodies.  Two observations have been pivotal. First, Chen and Alam with Harrison and Haynes have shown that both 2F5 and 4E10 require their respective heavy chain CDR3 hydrophobic loops to bind and neutralize virions.  Using surface plasmon resonance, they have shown that 2F5 and 4E10 mAbs do not bind to untriggered Env trimer gp41, but rather, only bind to virion lipids.  These data directly demonstrated two critical insights for vaccine design.  First, for the first time it has been definitively shown that the gp41 intermediate form of the MPER that is transiently expressed is indeed the optimal target of 2F5 and 4E10 mAbs. Second, these data demonstrated that lipids were an integral part of the nominal epitope of these two mabs, directly informing  HIV immunogen design.

 
Submitted September 22, 2008

Our goal is to acquire proof of concept data that manipulation of the immunoregulatory controls of B cell immune responses to HIV-1 Env, coupled with enhanced immunogen design, can lead to safe induction of broadly reactive neutralizing antibody responses. We are using a two armed approach to the problem of induction of antibodies that broadly neutralize HIV.

The Kelsoe, Haynes and Thorpe laboratories are developing immunogen formulations that trigger B cells normally tolerant to the desired Envelope epitopes and regions. Garnett Kelsoe is determining the origins, development, physiology, and fates of marginal zone, transitional and B1 B cell populations in animal models including mice and non-human primates. Haynes is determining the role of tolerance mechanisms, and TLR signaling on control of broadly reactive B cell activation, and Philip Thorpe is determining the role of lipid binding of anti-HIV antibodies and anti-phosphatidylserine (PS) autoantibodies to protection from HIV infection. The team has recently found that non-pathogenic anti-lipid antibodies that do not require beta-2-glycoprotein-1 for lipid binding do prevent HIV-1 and SHIVSF162P3 (and all R5 HIVs tested from infecting PBMC in vitro, and a prototype of non-pathogenic anti-lipid antibodies will be studied in vivo for the ability to protect against R5 SHIV infection.

The Harrison, Alam, Spicer, Shaw, Robinson and Hahn laboratories are developing immunogens that are in native conformations and are being tested for induction of neutralizing antibodies. These include HIV Env immunogens with a spectrum of affinities for binding to broadly reactive neutralizing antibodies, and immunogens with low entropic barriers to Mab binding and therefore are thermodynamically stable. Steve Harrison, Bing Chen and Larry Liao have made single chain Fv 4E10 antibodies and whole IgG1 2F5 Mabs with mutations that selectively eliminate gp41 or lipid reactivity. They have demonstrated that both 2F5 and 4E10 require the capacity to bind to lipids to neutralize HIV-1. They will now use the Fvs and Mabs in co-crystallization efforts with trimeric gp120/gp41 constructs. Munir Alam has designed gp41 peptide lipid conjugates using 4E10 and 2F5 epitopes peptides. He is characterizing the binding kinetics, and thermodynamic properties of 4E10 and 2F5 binding to peptide-lipid conjugates. Heather Desaire is characterizing the carbohydrates of HIV-1 Env produced in T cells and macrophages, and Haynes is determining methods for making these “autoantigens” immunogenic. Spicer is studying the lipid-peptide conjugates for their structures by NMR. George Shaw and Beatrice Hahn have constructed HIV-1/HIV-2 chimeras with HIV-2 neutralizing determinants in HIV-1 scaffolds, to study the neutralization of HIV-2 with James Robinson . The Team led by Victor Chalwe at the Tropical Disease Research Laboratory in Ndola, Zambia is providing the CAVD with PBMC and plasma on a series of chronically infected subjects for mapping epitopes of broadly neutralizing antibodies for immunogen design.

 

Submitted February 1, 2008 (Interim Report)

Our goal is to acquire proof of concept data that manipulation of the immunoregulatory controls of B cell immune responses to HIV-1 Env, coupled with enhanced immunogen design, can lead to safe induction of broadly reactive neutralizing antibody responses. We are using a two armed approach to the problem of induction of antibodies that broadly neutralize HIV.
The Kelsoe, Haynes and Thorpe laboratories are developing immunogen formulations that trigger B cells normally tolerant to the desired Envelope epitopes and regions. Garnett Kelsoe is determining the origins, development, physiology, and fates of marginal zone, transitional and B1 B cell populations in animal models including mice and non-human primates. Haynes is determining the role of tolerance mechanisms, and TLR signaling on control of broadly reactive B cell activation, and Philip Thorpe is determining the role of lipid binding of anti-HIV antibodies and anti-phosphatidylserine (PS) autoantibodies to protection from HIV infection. We have completed the first protection trial to determine if anti-beta-2-glycoprotein-1 antibodies can prevent infection or early viral destruction of the immune system in acute SIV infection.

The Harrison, Alam, Spicer, Shaw, Robinson and Hahn laboratories are developing immunogens that are more native and are being tested for induction of desired antibody types. These include HIV Env immunogens with a spectrum of affinities for binding to broadly reactive neutralizing antibodies, and immunogens with low entropic barriers to Mab binding and therefore are thermodynamically stable. Steve Harrison, Bing Chen and Larry Liao have made single chain Fv 4E10 antibodies and whole IgG1 2F5 Mabs with mutations that selectively eliminate gp41 or lipid reactivity. They have demonstrated that both 2F5 and 4E10 require the capacity to bind to lipids to neutralize HIV-1. They will now use the Fvs and Mabs in co-crystallization efforts with trimeric gp120/gp41 constructs. Munir Alam is designing gp41 peptide lipid conjugates using 4E10 and 2F5 epitopes peptides. He is characterizing the binding kinetics, and thermodynamic properties of 4E10 and 2F5 binding to peptide-lipid conjugates. Heather Desaire is characterizing the carbohydrates of HIV-1 Env produced in T cells and macrophages, and Haynes is determining methods for making these “autoantigens” immunogenic. Spicer is studying the lipid-peptide conjugates for their structures by NMR. George Shaw and Beatrice Hahn have constructed HIV-1/HIV-2 chimeras with HIV-2 neutralizing determinants in HIV-1 scaffolds, to study the neutralization of HIV-2 with James Robinson.

 

Submitted September 1, 2007

Our goal is to acquire proof of concept data that manipulation of the immunoregulatory controls of B cell immune responses to HIV-1 Env, coupled with enhanced immunogen design, can lead to safe induction of broadly reactive neutralizing antibody responses. We are using a two armed approach to the problem of induction of antibodies that broadly neutralize HIV.

The Kelsoe, Haynes and Thorpe laboratories are developing immunogen formulations that trigger B cells normally tolerant to the desired Envelope epitopes and regions. Garnett Kelsoe is determining the origins, development, physiology, and fates of marginal zone, transitional and B1 B cell populations in animal models including mice and non-human primates. Haynes is determining the role of tolerance mechanisms, and TLR signaling on control of broadly reactive B cell activation, and Philip Thorpe is determining the role of lipid binding of anti-HIV antibodies and anti-phosphatidylserine(PS) autoantibodies to protection from HIV infection. With Norm Letvin, a protection trial is being planned to determine if anti-PS antibodies can prevent infection or early viral destruction of the immune system in acute SIV infection.

The Harrison, Alam, Spicer, Shaw, Robinson and Hahn laboratories are developing immunogens that are more “native” and will preferentially induce the desired antibody types. These include HIV Env immunogens with a spectrum of affinities for binding to broadly reactive neutralizing antibodies, and immunogens with low entropic barriers to Mab binding and therefore are thermodynamically stable. Steve Harrison and Bing Chen are expressing and characterizing single-chain Fvs from 4E10, 2F5 and 2G12 Mabs to dissect lipid and protein contributions to binding and Mab neutralization. They will use the Fvs in co-crystallization efforts with trimeric gp120/gp41 constructs. Munir Alam is designing gp41 peptide lipid conjugates using 4E10 and 2F5 epitopes peptides. He is characterizing the binding kinetics, and thermodynamic properties of 4E10 and 2F5 binding to peptide-lipid conjugates. Desaire is characterizing the carbohydrates of HIV-1 Env produced in T cells and macrophages, and Haynes is determining methods for making these “autoantigens” immunogenic. Spicer is studying the lipid-peptide conjugates for their structures by NMR. George Shaw and Beatrice Hahn have constructed HIV-1/HIV-2 chimeras with HIV-2 neutralizing determinants in HIV-1 scaffolds, to study the neutralization of HIV-2.

 
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