Understanding the human antibody response to a malaria transmission-blocking vaccine
Lead Research Organisation:
University of Oxford
Department Name: The Jenner Institute
Abstract
Malaria still kills over 620,000 people a year in some of the poorest nations on earth, with over 75% being children. Malaria is an ancient and complicated parasite that is transmitted by bites of infected mosquitoes when they feed on human blood, hence much effort has been aimed directly as the mosquito itself with some success. Whilst there has been progress to reduce the death toll of this disease, progress is faltering and made worse by the prospect of insecticide resistant strains of mosquitoes and drug resistant parasites.
As has been demonstrated many times in the past and the recent Covid-19 pandemic, vaccines are one of the most effective weapons to combat any infectious disease. However despite huge efforts over the past 50 years progress has been limited, with only recently vaccines offering protection from life-threatening malaria infections. To improve the protection that vaccines can provide against malaria, the goal of the scientific community is to make a multi-component vaccine that targets the different life cycle stages of the parasite.
Malarias life cycle is complex, with an asexual stage in the human host and a sexual stage in the mosquito midgut. When a mosquito feeds on a malaria infected person, they transmit the parasite to another uninfected human. Transmission blocking vaccines (TBVs) work by generating antibodies in the human host, that when a mosquito feeds on a malaria infected person, are taken up with the bloodmeal into the mosquito midgut. These antibodies find their molecular targets and latch onto proteins that only reveal themselves in the mosquito midgut, preventing these proteins from performing their function and preventing the parasite from completing it's life cycle. This leaves the mosquito unable to transmit malaria to the next individual.
One of the leading malaria TBV candidates is a protein called Pfs48/45 which shows excellent results in pre-clinical studies and a human clinical trial is scheduled to start in May 2022. In this project, we will understand the antibody response generated against Pfs48/45 in vaccinated volunteers and use this information to help design better vaccines. This will significantly enhance our understanding of the human immune response to Pfs48/45, as most of our knowledge is currently based on pre-clinical studies, which might not be representative of how the human immune system would react and which areas of Pfs48/45 human antibodies might target.
We will isolate antibody producing cells from Pfs48/45 vaccinated clinical trial volunteers and from them extract the genetic information coding for antibodies that target Pfs48/45, allowing us to easily produce them in the lab. We will then study these antibodies, test how good they are at blocking transmission of the malaria parasite in a pre-clinical model, find out how strongly they bind to, and where on, Pfs48/45 they bind. This will allow us to define what makes a good blocking antibody and which regions of Pfs48/45 are useful to target.
We will also look at the total antibody response in the sera of volunteers, seeing which regions of Pfs48/45 are targeted, and whether this changes as volunteers receive multiple doses or different doses of the vaccine.
Using advances in computer modelling of proteins, we will also make versions of Pfs48/45 that can be produced with reduced cost and are more stable, an important consideration for any vaccine to be deployed in Africa. We will test the stability of these proteins and their ability to be produced in a simple cell expression systems.
This research will help build our understanding of the human antibody response to Pfs48/45 and use this information to design improved vaccines. This combined with making more stable versions of Pfs48/45 will lay the foundation for the next generation of Pfs48/45 vaccines.
As has been demonstrated many times in the past and the recent Covid-19 pandemic, vaccines are one of the most effective weapons to combat any infectious disease. However despite huge efforts over the past 50 years progress has been limited, with only recently vaccines offering protection from life-threatening malaria infections. To improve the protection that vaccines can provide against malaria, the goal of the scientific community is to make a multi-component vaccine that targets the different life cycle stages of the parasite.
Malarias life cycle is complex, with an asexual stage in the human host and a sexual stage in the mosquito midgut. When a mosquito feeds on a malaria infected person, they transmit the parasite to another uninfected human. Transmission blocking vaccines (TBVs) work by generating antibodies in the human host, that when a mosquito feeds on a malaria infected person, are taken up with the bloodmeal into the mosquito midgut. These antibodies find their molecular targets and latch onto proteins that only reveal themselves in the mosquito midgut, preventing these proteins from performing their function and preventing the parasite from completing it's life cycle. This leaves the mosquito unable to transmit malaria to the next individual.
One of the leading malaria TBV candidates is a protein called Pfs48/45 which shows excellent results in pre-clinical studies and a human clinical trial is scheduled to start in May 2022. In this project, we will understand the antibody response generated against Pfs48/45 in vaccinated volunteers and use this information to help design better vaccines. This will significantly enhance our understanding of the human immune response to Pfs48/45, as most of our knowledge is currently based on pre-clinical studies, which might not be representative of how the human immune system would react and which areas of Pfs48/45 human antibodies might target.
We will isolate antibody producing cells from Pfs48/45 vaccinated clinical trial volunteers and from them extract the genetic information coding for antibodies that target Pfs48/45, allowing us to easily produce them in the lab. We will then study these antibodies, test how good they are at blocking transmission of the malaria parasite in a pre-clinical model, find out how strongly they bind to, and where on, Pfs48/45 they bind. This will allow us to define what makes a good blocking antibody and which regions of Pfs48/45 are useful to target.
We will also look at the total antibody response in the sera of volunteers, seeing which regions of Pfs48/45 are targeted, and whether this changes as volunteers receive multiple doses or different doses of the vaccine.
Using advances in computer modelling of proteins, we will also make versions of Pfs48/45 that can be produced with reduced cost and are more stable, an important consideration for any vaccine to be deployed in Africa. We will test the stability of these proteins and their ability to be produced in a simple cell expression systems.
This research will help build our understanding of the human antibody response to Pfs48/45 and use this information to design improved vaccines. This combined with making more stable versions of Pfs48/45 will lay the foundation for the next generation of Pfs48/45 vaccines.
Technical Summary
90 monoclonal antibodies will be cloned from human PBMCs. B cells will be isolated using fluorescent-sorting, gating using a B cell marker and fluorescently labelled Pfs48/45-containing tetramers. IgG variable regions will be amplified by RT-PCR and sequenced. Heavy and light chain V(D)Js will be inserted into Fc-containing plasmids, expressed in HEK293 cells and purified by protein G chromatography.
The standard membrane feeding assay will be used to evaluate the ability of sera and the monoclonal antibodies to block transmission of P. falciparum . Stage V gametocytes will be mixed with purified monoclonal antibodies or IgG from serum samples and then fed to 4-6 days old starved female Anopheles stephensi (SDA 500) mosquitoes. After 8 days, midguts from 20 mosquitoes per group will be dissected, oocysts counted, and the number of infected mosquitoes recorded.
SPR will be conducted on a Carterra LSA platform, capable of immobilising 384 samples for high throughput determination of binding kinetics and competition.
Electron microscopy polyclonal epitope mapping will use polyclonal sera, from which Fab fragments will be produced by enzymatic cleavage. These will be incubated with Pfs48/45 already complexed with mAbs of known epitope as markers. Complexes will be purified by size exclusion chromatography and structures determined by negative stain electron microscopy and single particle analysis.
To determine crystal structures, Fab fragments will be produced by papain cleavage and bound to Pfs48/45. Complexes will be assembled and purified by size exclusion chromatography and crystallised. Synchrotron data will be used to determine structures, with phase information derived by molecular replacement, using our existing Pfs48/5 structures.
To thermally stabilise Pfs48/45, we will use Rosetta-based protein design tools, employed through the PROSS methodology. Designs will be selected to increase domain stability and to lock together domain interfaces.
The standard membrane feeding assay will be used to evaluate the ability of sera and the monoclonal antibodies to block transmission of P. falciparum . Stage V gametocytes will be mixed with purified monoclonal antibodies or IgG from serum samples and then fed to 4-6 days old starved female Anopheles stephensi (SDA 500) mosquitoes. After 8 days, midguts from 20 mosquitoes per group will be dissected, oocysts counted, and the number of infected mosquitoes recorded.
SPR will be conducted on a Carterra LSA platform, capable of immobilising 384 samples for high throughput determination of binding kinetics and competition.
Electron microscopy polyclonal epitope mapping will use polyclonal sera, from which Fab fragments will be produced by enzymatic cleavage. These will be incubated with Pfs48/45 already complexed with mAbs of known epitope as markers. Complexes will be purified by size exclusion chromatography and structures determined by negative stain electron microscopy and single particle analysis.
To determine crystal structures, Fab fragments will be produced by papain cleavage and bound to Pfs48/45. Complexes will be assembled and purified by size exclusion chromatography and crystallised. Synchrotron data will be used to determine structures, with phase information derived by molecular replacement, using our existing Pfs48/5 structures.
To thermally stabilise Pfs48/45, we will use Rosetta-based protein design tools, employed through the PROSS methodology. Designs will be selected to increase domain stability and to lock together domain interfaces.