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Periodic Report Summary 1 - RATIONAL HIV VACCINE (Rational HIV vaccine design via mammalian cell display)

Project No: 623038

Project Acronym: Rational HIV Vaccine
Project Full Name: Rational HIV vaccine design via mammalian cell display

AIMS: This application proposes experiments to investigate one of the greatest challenges in HIV vaccine research: how can broadly neutralising antibodies be induced by vaccination in humans to target pathogens such as HIV that have been resistant to traditional vaccination approaches?

AIM [i] Identification of HIV antigens that bind germ line and matured broadly neutralising antibodies

The major project objectives for the first two years of this project regarding Aim [i] were to produce a panel of antigen variants and test them for binding to neutralising and germ line antibodies of this bNAb family and assess their ability to stimulate B cells expressing precursor antibodies.

AIM [ii] Optimisation of antigens to favour elicitation of broadly neutralising antibodies

The major project objectives for the first two years of this project regarding AIM [ii] were to generate at least one combinatorial antigen library for mammalian display and select antigens with higher affinity for germ line and mature bNAbs with this method. In addition, training objectives were to achieve competence in flow cytometry-based cell sorting and antigen mutagenesis protocols.

AIM [iii] Using selected antigens to mature germ line antibodies to a broadly neutralising phenotype

The projected objectives for the outgoing phase (year 1 and 2) for AIM [iii] were to validate antigen-dependent magnetic bead assay and FACS sorting protocols with cells expressing mature neutralising antibodies, and then to establish a system to enrich populations of cells with higher affinity for HIV antigen and then sequence the antibodies encoded and verify the increase of affinity.

WORK COMPLETED TO DATE
A panel of antigen variants based on HIV envelope outer domain, gp120 and gp140 proteins were tested for the ability to bind to broadly neutralising N332-specific PGT121 family antibodies and their inferred germ line precursors. As no detectable binding activity was seen to germ line antibodies, the least mutated antibodies found to have anti-HIV activity were used to select for optimised germ line binding antigens. In collaboration with the International AIDS Vaccine Initiative at The Scripps Research Institute, this was achieved using mammalian display stable cells lines expressing the following: an optimised HIV envelope protein outer domain, an error-prone PCR random mutagenesis HIV envelope gp120 library, a structurally guided NNK degenerate codon HIV envelope gp140 library and an NNK scanning HIV envelope gp120 library. Antigen-dependent magnetic bead assays and flow cytometry sorting protocols were validated and the latter found to be more efficient at selecting high affinity cell surface interactions than bead-based selection. Consequently, the mammalian display libraries were enriched by multiple rounds of FACS cell sorting to yield antigens with improved binding ability for germ line antibodies.

Simultaneously, lentiviral systems were used to generate membrane-anchored antibody-expressing human cell lines with both broadly neutralising precursor antibodies and antibody libraries with inherent sequence variation. Transient transfection of the enzyme responsible for natural antibody mutation, activation-induced cytidine deaminase (AID), resulted in mutation of transgenes but in a non-targeted manner that caused significant disruption to the functioning of the cells and did not result in enough antibody gene mutation. Cell lines derived from human B cells spontaneously use AID to hypermutate within the variable regions of the antibody gene locus but we found they could not efficiently mutate transgenes introduced randomly into the genome. This is thought to be due to the presence of regulatory factors in the B cells. Therefore to enable in vitro maturation of HIV-specific antibodies in these cells it is necessary to alter the antibody genetic locus to swap the cell’s original antibody gene for those encoding anti-HIV antibodies. This was achieved using CrispR-cas9 technology to insert the heavy chain variable region of the anti-HIV antibody PG9 into the heavy chain antibody locus. This produced cells expressing functional chimeric antibody with the B cells own light chain on their surface. As these cells continue to hypermutate with cell division they will be selected by FACS for improved binding to targeted HIV antigens which will facilitate in vitro affinity maturation of the chimeric antibody.

SIGNIFICANT RESULTS
• Directed evolution of PGT121 germ line binding antigens via mammalian display which stimulate B cells encoding germ line antibodies[1,2]
• Generation of cell lines expressing bNAb precursor and germ line antibodies including endogenous locus replacement system to enable in vitro maturation[3,4]
• Validation of B cell stimulatory activity via collaborators follow on in vivo immunogenicity study[5]

[1] Keystone HIV Vaccines meeting 2016: Steichen JM, McCoy LE, Kulp DW et al.
[2] Steichen et al. Immunity, 2016. 45: p. 1–14.
[3] Andrabi et al. Immunity. 2015 Nov 17;43(5):959-73.
[4] Cell and Gene Therapy for HIV Cure 2016: Voss JE, Andrabi R, McCoy LE et al.
[5] Escolano et al. Cell, 2016. 166(6): p. 1445-1458 e12

FINAL GOALS AND POTENTIAL IMPACT
• Validation of immune stimulation capacity of germline binding antigens which will enable their development as immunogens
• In vitro maturation of an HIV neutralizing antibody which will allow faster iteration of immunogen candidates and the development of potential prophylactics and therapeutics

Reported by

UNIVERSITY COLLEGE LONDON
United Kingdom

Subjects

Life Sciences
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