Development of llama single-domain antibodies as ingredient for an HIV -1 entry-inhibitor microbicide (2024)

Gorlani, A. (2012). Development of llama single-domain antibodies as ingredient for an HIV -1 entry-inhibitor microbicide. [Doctoral thesis 1 (Research UU / Graduation UU), Utrecht University]. Utrecht University.

Gorlani, A.. / Development of llama single-domain antibodies as ingredient for an HIV -1 entry-inhibitor microbicide. Utrecht : Utrecht University, 2012. 165 p.

@phdthesis{ce9ede0f6bda4e15b0157b1e9da98b44,

title = "Development of llama single-domain antibodies as ingredient for an HIV -1 entry-inhibitor microbicide",

abstract = "Heavy-chain antibodies are present in llamas next to conventional immunoglobulins. Their variable domain, also known as VHH is a small, single-chain globular protein with full antigen-binding capacity. Its single-domain nature and some structural differences from conventional immunoglobulins make it a tool with high potential for a number of biotechnological and medical applications. The HIV pandemic represents since three decades a dramatic health problem throughout the world and especially in resource-limited countries. As an alternative to vaccination, prevention is regarded as the best way to control the pandemic, both from a healthcare and economic point of view. The development of microbicides that help preventing sexually transmitted infections of HIV (the most common way the virus is transmitted) is therefore highly desirable. In this thesis, several aspects of the development of llama heavy-chain antibody fragments as active compound of topical microbicides are investigated. In chapter one the 3D structure of the HIV neutralizing VHH D7 was determined. The extension, flexibility and amino acid-composition of its CDRs were compared to other known (human) neutralizing antibodies in order to define the way D7 neutralizes the virus by binding gp120. Structural comparison suggested diverse modes of interaction. Mutational analysis identified CDR3 as key area of the interaction with gp120, whose conformational flexibility is likely to accommodate multiple modes of antibody binding inside the CD4 binding site. Comparison of the related VHH A12 and D7 revealed broad and narrow cross-clade neutralization phenotypes respectively. In chapter two a family-specific phage library was generated and a number of hom*ologous VHH was selected with varying neutralizing profiles. Analysis of their sequences and their recognition of a panel of different HIV-1 subtypes, allowed the understanding of the molecular basis of the different neutralization behaviors. In particular the important role played by the last two C-terminal amino acids of CDR3 was demonstrated. Secretion efficiency of VHH is a particularly important factor when large amounts are needed at low production cost. In chapter three we analyzed databases of structures and sequences to find sequence-related factors that influence the yield of VHH when produced in the host S. cerevisiae. We showed how the combined presence of five key residues and specific J segments is fundamental for proper folding. We also suggested that interactions with ER-resident chaperones are necessary to achieve high secretion efficiency. In the last chapter, VHH A12 and D7 were used as models to study their availability, activity and stability in a VHH-based topical microbicide. We systematically analyzed likely challenges that VHH will have to face in case of use in sub-tropical settings of resource-limited countries, as HIV preventative drug. VHH proved to be readily available when formulated as tablets or aqueous gel and very stable after extended incubation at physiological temperature and at low pH. We also investigated the tissue-penetration kinetics of VHH in order to assess whether the drug would be present in the sub-mucosal layers of the vagin* and once there, carry out its protective action.",

author = "A. Gorlani",

year = "2012",

month = jan,

day = "9",

language = "Undefined/Unknown",

publisher = "Utrecht University",

type = "Doctoral thesis 1 (Research UU / Graduation UU)",

school = "Utrecht University",

}

Gorlani, A 2012, 'Development of llama single-domain antibodies as ingredient for an HIV -1 entry-inhibitor microbicide', Doctor of Philosophy, Utrecht University, Utrecht.

Development of llama single-domain antibodies as ingredient for an HIV -1 entry-inhibitor microbicide. / Gorlani, A.
Utrecht: Utrecht University, 2012. 165 p.

Research output: Thesis › Doctoral thesis 1 (Research UU / Graduation UU)

TY - THES

T1 - Development of llama single-domain antibodies as ingredient for an HIV -1 entry-inhibitor microbicide

AU - Gorlani, A.

PY - 2012/1/9

Y1 - 2012/1/9

N2 - Heavy-chain antibodies are present in llamas next to conventional immunoglobulins. Their variable domain, also known as VHH is a small, single-chain globular protein with full antigen-binding capacity. Its single-domain nature and some structural differences from conventional immunoglobulins make it a tool with high potential for a number of biotechnological and medical applications.The HIV pandemic represents since three decades a dramatic health problem throughout the world and especially in resource-limited countries. As an alternative to vaccination, prevention is regarded as the best way to control the pandemic, both from a healthcare and economic point of view. The development of microbicides that help preventing sexually transmitted infections of HIV (the most common way the virus is transmitted) is therefore highly desirable. In this thesis, several aspects of the development of llama heavy-chain antibody fragments as active compound of topical microbicides are investigated.In chapter one the 3D structure of the HIV neutralizing VHH D7 was determined. The extension, flexibility and amino acid-composition of its CDRs were compared to other known (human) neutralizing antibodies in order to define the way D7 neutralizes the virus by binding gp120. Structural comparison suggested diverse modes of interaction. Mutational analysis identified CDR3 as key area of the interaction with gp120, whose conformational flexibility is likely to accommodate multiple modes of antibody binding inside the CD4 binding site.Comparison of the related VHH A12 and D7 revealed broad and narrow cross-clade neutralization phenotypes respectively. In chapter two a family-specific phage library was generated and a number of hom*ologous VHH was selected with varying neutralizing profiles. Analysis of their sequences and their recognition of a panel of different HIV-1 subtypes, allowed the understanding of the molecular basis of the different neutralization behaviors. In particular the important role played by the last two C-terminal amino acids of CDR3 was demonstrated.Secretion efficiency of VHH is a particularly important factor when large amounts are needed at low production cost. In chapter three we analyzed databases of structures and sequences to find sequence-related factors that influence the yield of VHH when produced in the host S. cerevisiae. We showed how the combined presence of five key residues and specific J segments is fundamental for proper folding. We also suggested that interactions with ER-resident chaperones are necessary to achieve high secretion efficiency.In the last chapter, VHH A12 and D7 were used as models to study their availability, activity and stability in a VHH-based topical microbicide. We systematically analyzed likely challenges that VHH will have to face in case of use in sub-tropical settings of resource-limited countries, as HIV preventative drug. VHH proved to be readily available when formulated as tablets or aqueous gel and very stable after extended incubation at physiological temperature and at low pH. We also investigated the tissue-penetration kinetics of VHH in order to assess whether the drug would be present in the sub-mucosal layers of the vagin* and once there, carry out its protective action.

AB - Heavy-chain antibodies are present in llamas next to conventional immunoglobulins. Their variable domain, also known as VHH is a small, single-chain globular protein with full antigen-binding capacity. Its single-domain nature and some structural differences from conventional immunoglobulins make it a tool with high potential for a number of biotechnological and medical applications.The HIV pandemic represents since three decades a dramatic health problem throughout the world and especially in resource-limited countries. As an alternative to vaccination, prevention is regarded as the best way to control the pandemic, both from a healthcare and economic point of view. The development of microbicides that help preventing sexually transmitted infections of HIV (the most common way the virus is transmitted) is therefore highly desirable. In this thesis, several aspects of the development of llama heavy-chain antibody fragments as active compound of topical microbicides are investigated.In chapter one the 3D structure of the HIV neutralizing VHH D7 was determined. The extension, flexibility and amino acid-composition of its CDRs were compared to other known (human) neutralizing antibodies in order to define the way D7 neutralizes the virus by binding gp120. Structural comparison suggested diverse modes of interaction. Mutational analysis identified CDR3 as key area of the interaction with gp120, whose conformational flexibility is likely to accommodate multiple modes of antibody binding inside the CD4 binding site.Comparison of the related VHH A12 and D7 revealed broad and narrow cross-clade neutralization phenotypes respectively. In chapter two a family-specific phage library was generated and a number of hom*ologous VHH was selected with varying neutralizing profiles. Analysis of their sequences and their recognition of a panel of different HIV-1 subtypes, allowed the understanding of the molecular basis of the different neutralization behaviors. In particular the important role played by the last two C-terminal amino acids of CDR3 was demonstrated.Secretion efficiency of VHH is a particularly important factor when large amounts are needed at low production cost. In chapter three we analyzed databases of structures and sequences to find sequence-related factors that influence the yield of VHH when produced in the host S. cerevisiae. We showed how the combined presence of five key residues and specific J segments is fundamental for proper folding. We also suggested that interactions with ER-resident chaperones are necessary to achieve high secretion efficiency.In the last chapter, VHH A12 and D7 were used as models to study their availability, activity and stability in a VHH-based topical microbicide. We systematically analyzed likely challenges that VHH will have to face in case of use in sub-tropical settings of resource-limited countries, as HIV preventative drug. VHH proved to be readily available when formulated as tablets or aqueous gel and very stable after extended incubation at physiological temperature and at low pH. We also investigated the tissue-penetration kinetics of VHH in order to assess whether the drug would be present in the sub-mucosal layers of the vagin* and once there, carry out its protective action.

M3 - Doctoral thesis 1 (Research UU / Graduation UU)

PB - Utrecht University

CY - Utrecht

ER -

Gorlani A. Development of llama single-domain antibodies as ingredient for an HIV -1 entry-inhibitor microbicide. Utrecht: Utrecht University, 2012. 165 p.