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Weiss Progress Report Abstracts continued

 
Submitted August 1, 2011

The UCL VDC contributes towards the CAVD’s understanding of humoral immunity to HIV infection with the aim of devising immunogens that will elicit neutralizing antibodies in vaccinated individuals. This project has entailed the isolation of novel potently neutralizing monoclonal antibodies (MAbs) from infected persons and from immunized llamas and mice, the selection of novel peptide and protein immunogens recognized by such MAbs, and testing whether they elicit the production neutralizing immunity in animals as a prelude to eventual clinical trial. Our general progress during the past year has been to show that one of the novel human MAbs can completely block infection of in macaques of a hybrid SHIV bearing a subtype C HIV envelope in a trial of passive immuniation. Promising results in generating neutralizing antibodies in rabbit and guinea-pigs immunized with our antigens will be taken forward to tests of improved combinations of antigens and adjuvants in macaques followed by challenge by SHIV in order to determine parameters of protection from infection. In addition, highly potent and broadly neutralizing llama single-chain antibody fragments have been characterized functionally and structurally in its interaction with the HIV envelope glycoprotein, and have the potential to be developed as protective vaginal microbicides. The isolation of potent llama antibodies also demonstrates that immunization with improved HIV enveloped proteins can elicit neutralizing antibodies similar to those rare antibodies found in elite neutralizers in long-term HIV-infected persons, and thus gives much promise for vaccine development.

 
Submitted January 14, 2011 (Interim Report)

The main objective of this vaccine Consortium is to gain greater insight into and develop reagents for protection against HIV-1 infection by neutralizing antibodies (humoral immunity). Four specified aims are:

  1. 1.  Identify novel neutralizing monoclonal antibodies (NMAbs) that cross-neutralize many strains of HIV-1 between and within clades.
  2. 2. Demonstrate that some of these NMAbs can protect against mucosal infection by humanized SIV (SHIV clade C) by passive transfer to macaques.
  3. 3. Direct the selection of novel immunogens recognized by these NMAbs. These novel immunogens serve as the basis for the development of HIV-1 vaccine candidates for humoral immunity.
  4. 4.  Demonstrate the ability of the vaccine candidates to elicit anti-HIV-1 antibodies in animal models.

 Successful candidates from this Vaccine Discovery Group may progress to testing in clinical trials later.

Scientific achievements since February 2010:

Novel NMAbs:

  • We isolated 98 MAbs from memory B cells of HIV-1 infected patients recruited in Antwerp & London of all major clades and circulating recombinant forms of HIV-1;
  • We isolated over 200 NMabs from llamas which showed a different neutralization profiles including broad ones, and recognized epitopes in the CD4 binding site, a CD4-induced epitope (CD4i) and gp41 including MPER.
  • We isolated 69 human IgM monoclonals specific for gp140 from the HuIg mice, including some that neutralize as IgM but curiously are non-neutralizing when converted to IgG.

 Antigens:

  • We developed a novel Ag screening method using lentivirus display technology tested first with known NMAbs and then with our novel NMAbs.
  • We developed novel, improved epitopes and mimotopes on peptide scaffolds which were tested as antigens.
  • We developed novel VLPs and MAb derived Env structures which were tested as antigens.
  • We characterized 5 different gp41 antigens which are under test as immunogens in rabbits.

Protection:

  • We successfully conducted passive protection of macaques from mucosal challenge with a Clade C SHIV by a single anti-HIV-1 V3 loop NMAB, HGN194.
Submitted July 26, 2010

This project aims to discover vaccine components that induce long-lasting neutralizing antibodies to HIV-1.

We have characterised and mapped 3 novel broadly neutralizing human monoclonal antibodies (NMAbs) (Corti et al, PLOS one, 2010) and generated antigenic structures encoding their epitope specificity - (V3, CD4 binding site and gp41 HR1). These NMAb are currently in crystallographic analysis in complex with their respective antigens. These 3 MAbs have been scaled-up under GLP production, and one of these HNG194 has been evaluated in vivo in Rhesus macaques to demonstrate passive protection from SHIV challenge. VHH from immunized llamas have been comprehensively characterized for neutralizing activity and epitope specificity and selected members of this reagent cluster are undergoing crystallographic analysis. One NMAb isolated from the HuIg mouse model is broadly reactive with HIV-1 Clade A and C gp120 V3 loops, and is has been produced in recombinant IgG form to complete full-scale neutralization and epitope analysis. Several other novel MAbs cloned from the HuIg mice show neutralizing activity and are currently being produced in recombinant IgG form and characterized for breadth of neutralization and epitope specificity. Lastly, we have taken the strategic decision, with support from our SAB, to continue production of human MAb with specifically selected patients using optimized NAb screening assays.

Based on our new NMAbs and pre-existing ones, we have designed peptide-based antigens, and evaluated presentation systems such as virus-like particles, and trimeric recombinant gp140 protein antigens as immunogens to elicit neutralizing Abs in rabbits, rats and Guinea pigs. Various adjuvant systems have been evaluated for B-cell immunogenicity in conjunction with these immunogens. We are now in the iterative process of evaluating the optimal antigen-adjuvant formulations to take forward for immunization and challenge studies in macaques.

 
Submitted January 14, 2010 (Interim Report)

Research objectives for our consortium:
1. Identify novel monoclonal antibodies that cross-neutralize HIV-1 between and within Clades.
2. Direct the selection of novel immunogens recognized by these monoclonal antibodies. These novel immunogens will serve as the basis for the creation of HIV-1 vaccine candidates.
3. Demonstrate the ability of these novel immunogen vaccine candidates to elicit anti-HIV-1 antibodies in animal models. Successful candidates may progress to testing in clinical trials.

Scientific achievements since February 2008:
• Isolated 98 MAbs from memory B cells of HIV-1 infected recruited in Antwerp & London

• Isolated over 50 NMabs from llamas which showed a broad neutralization profile and recognized epitopes in the CD4 binding site, a CD4-induced epitope (CD4i) and gp41 including MPER

• Isolated 69 human IgM monoclonals specific for gp140 from the HuIg mice, including F44, which is of particular interest

• Developed novel Ag screening methods tested with known NMAbs

• Developed novel, improved epitopes and mimotopes on scaffolds which were tested as antigens

• Developed novel VLPs and MAb derived novel Env structures which were tested as antigens

• Characterised 5 different gp41 antigens which were provided for rabbit immunization studies. Structural studies of other NMAbs are ongoing.

• Initiated macaque studies with novel NMAb showing successful post challenge protection.

 
Submitted July 28, 2009

This project aims to discover vaccine components that induce long-lasting neutralizing antibodies to HIV-1. We have identified 3 novel broadly neutralizing human monoclonal antibodies (NMAbs) which have been comprehensively characterized for epitope specificity - mapped to gp120 V3 and CD4 binding site and gp41 HR1 - and neutralization activity. These NMAb are currently in crystallographic analysis in complex with their respective antigens, and a manuscript is submitted for publication. These 3 NMAbs are in the process of large-scale GLP production for use in in vivo macaque passive infusion studies, to evaluate their ability to protect macaques from SHIV challenge. VHH from immunized llamas have been comprehensively characterized for neutralizing activity and epitope specificity and selected members of this reagent cluster are undergoing crystallographic analysis. One NMAb isolated from the HuIg mouse model is broadly reactive with HIV-1 Clade A and C gp120 V3 loops, and is has been produced in recombinant IgG form to complete full-scale neutralization and epitope analysis. Several other novel MAbs cloned from the HuIg mice show neutralizing activity and are currently being produced in recombinant IgG form and characterized for breadth of neutralization and epitope specificity.

Based on our new NMAbs and pre-existing ones, we have designed peptide-based antigens, constructs on virus-like particles, and trimeric recombinant gp140 protein antigens for testing as immunogens eliciting neutralizing Abs in rabbits and rats. Various adjuvants have been tested for efficacy in conjunction with these immunogens. We shall select the best antigen-adjuvant formulations to take forward for immunization and challenge studies in macaques.

 
Submitted January 15, 2009 (Interim Report)

A successful HIV-1 vaccine probably needs to induce both antibody and cellular immunity. A starting point for antibody-based vaccine design is the identification of conserved antibody neutralization epitopes. At the start of the CAVD in 2006, only 5 neutralizing monoclonal antibodies (NMAbs) that broadly cross-neutralized multiple HIV-1 strains were known.

We therefore sought in Activity 1 to make more NMAbs that act against a spectrum of African HIV-1 strains. Of a total 58 MAbs isolated from HIV-1-infected humans, 25 cross-neutralize several HIV-1 strains, and 5 of these which have different properties from previously described NMAbs have been selected for more intensive study. In addition to the NMAbs isolated from infected people, we have successfully isolated NMAbs from llamas and genetically engineered HuIg mice that have been immunized with HIV-1 Env cloned from African HIV-1 isolates. HuIg mice produce antibodies with features of human immunoglobulins that may be important in HIV-1 neutralization, and llamas produce heavy chain-based NMAbs which provide properties that will be useful in screening and characterizing candidate components of an HIV-1 vaccine.

In Activity 2, we are using the NMAbs to identify protein and peptide structures that are derived from, or mimic epitopes on the HIV-1 envelope as candidate components of a vaccine. By re-iterative screening of slightly different molecules the aim is to identify ones that can be used to immunize individuals so as to generate protective Abs in the body. This is a long and detailed process that involves understanding the biochemistry, structural biology and stability of the molecules. In parallel we are exploring other novel strategies for designing Env-based antigens that are not reliant on antibody-based ‘reverse vaccinology’.

In Activity 3, the selected molecules (‘immunogens’) are being tested for their ability to induce NAbs in animals to evaluate which immunogens might be useful to take forward into preliminary clinical trials in humans. First, the immunogens are tested in rabbits and subsequently the best candidates will be tested in macaques which can be challenged with a live virus bearing an HIV-1 Env to determine whether the Abs actually protect against infection. Human studies will not be conducted during this grant period but we would aim to take our best immunogens further into clinical trials with subsequent CAVD funding for future studies. In addition to studying the immunogen itself, novel ‘adjuvants’ (accompanying molecules that stimulate immune responses) are being evaluated systematically for their efficacy with HIV-1 immunogens.

 
Submitted August 1, 2008

This project aims to discover vaccine components that induce long-lasting neutralizing antibodies (NAbs) to HIV-1, and will block infection in a person exposed to HIV-1. The specific objective is to develop molecules (immunogens) that cross-neutralize HIV-1 of Clades (subtypes) C, A and AG, which together comprise 75% of new HIV-1 infections occurring in Africa today.

The novel immunogens will be identified using unique monoclonal antibodies (MAb) which we will produce and characterise in Activity 1 of the program. These mAbs will be screened for their ability to neutralise a broad array of HIV-1 variants from multiple Clades, with the emphasis on Clades A/G and C. We have isolated cross-reactive NAbs from HIV-infected donors, and from immunized llamas which have unusual antibodies that lend themselves to high throughput screening; other NAbs from genetically modified mice are in the pipeline. We have more than 40 novel monoclonal NAbs that are in the process of extensive characterisation. A select number of these will be taken to activity 2 for the process of identifying novel immunogens. Our antibodies will also be made available to other HIV vaccine research groups.

In Activity 2 we have devised reiterative techniques to screen thousands, if not millions, of molecules to identify those that bind to these unique broadly neutralising NAbs. One approach will involve the use of chemically synthesized peptides related to pieces of the HIV envelope are attached to “scaffolds” that hold them in a configuration for optimal presentation to the immune system. We are also generating variants of envelope proteins on non-infectious virus-like particles by a method called gene-shuffling. Selected immunogens will then be tested in Activity 3 for their ability to elicit NAbs in animal models. If the immunized animals can be protected from challenge infection by viruses with Clade C or A/AG envelopes, our project will have been successfully concluded, and the immunogens will be available as components of vaccines in human clinical trials.

 
Submitted January 11, 2008 (Interim Report)

This VDC is based on a reverse vaccinology approach to identify immunogens that will elicit cross-protective neutralizing antibodies to HIV-1. Activity 1 aims to identify and provide novel neutralizing monoclonal antibodies (NMAbs) to substantially increase the number available to the AIDS vaccine research community. Activity 2 aims to use existing NMAbs and the new NMAbs to interrogate antigenic structures based on peptides and on env-based proteins to identify and select candidate immunogens that might become components of vaccines. Activity 3 aims to test candidate immunogens in vivo to select those that elicit cross-protective neutralizing antibody responses.

Milestones:

Identify up to 4 Clade C and 4 A/CRF02_AG patients with broad Nabs and provide B-memory cells for immortalization

Provide 5 NMAbs to Activity 2

Provide 5 more NMAbs to Activity 2

Select NMAbs to gp41 and gp120 to make and analyze Ag:Ab structures

Demonstrate initial selection of Ags with known NMAbs to clade B

Select Ags with first 5 NMAbs to Clade C and CRF02_AG

Provide 2 to 4 promising Ags to Activity 3 for pilot immunogenicity testing

Provide Ags to begin Ag:Ab structural analysis

Scientific achievements since February 2007

Institute for Research in Biomedicine, Switzerland

MAbs from memory B cells of HIV-1 infected patients in Antwerp & London

9 NMAbs, 2 broad & potent

University College London and University of Utrecht

Llama VHH, 38 NMAbs to CD4bs, in 2 families from CN54 gp120

6 new VHH from ZM96/UG37 gp140, not yet mapped

Medical Research Council Laboratory of Molecular Biology and University of Oxford

Mice transgenic for human Ig, under selection

Overall achievements

Novel NMAbs developed from human patients

Novel NMAbs obtained from llama VHH phage libraries

Novel NMAbs in pipeline from HuIg transgenic mice

Novel Ag screening methods tested with known NMAbs

Use of ZM96 trimeric gp140 as subtype C immunogen

Novel adjuvants for B-cell response in rabbits

Structural studies of new NMAbs initiated

ELISA screening SOPs based on Eurovac SOPs

Neutralization assays based on Duke VIMC SOPS

 
Submitted August 1, 2007

Our objective is to discover vaccine components that induce long-lasting neutralizing antibodies (Nabs) to HIV-1, and will block infection in a person exposed to HIV-1. The aim is to develop molecules (immunogens) that cross-neutralize HIV-1 of Clades (subtypes) C, A and AG, which together comprise 75% of new HIV-1 infections occurring in Africa today. 

In Activity 1 we are making novel monoclonal Nabs to screen for suitable immunogens. We have isolated cross-reactive Nabs from HIV-infected donors, and from immunized llamas which have unusual antibodies that lend themselves to high throughput screening; other Nabs from genetically modified mice are in the pipeline. Our monoclonal Nabs will also be useful to other HIV vaccine research groups.

In Activity 2 we have devised reiterative techniques to screen thousands, if not millions, of molecules to identify those that bind to the Nabs. Chemically synthesized peptides related to pieces of the HIV envelope are attached to “scaffolds” that hold them in a configuration for optimal presentation to the immune system. We are also generating variants of envelope proteins on non-infectious virus-like particles by a method called gene-shuffling. Selected immunogens will then be tested in Activity 3, for their ability to elicit Nabs, initially in rabbits, and for the most promising ones, in macaques. If the immunized macaques are protected from challenge infection by viruses with Clade C or A/AG envelopes, our project will have been successfully concluded, and the immunogens can then be tested as components of vaccines in human clinical trials.

 
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