Genetically attenuated malaria parasites (GAP) as a vaccine

What is the current status of a GAP-based vaccine and future directions?

  • As part of a TI-Pharma funded project the LUMC, RadboudMC and the American company, SANARIA, created a genetically attenuated parasite (GAP; Pf∆slarpb9).  
  • This human GAP and the equivalent rodent GAP has been evaluated using a set of preclinical safety and efficacy studies
  • SANARIA has generated an aseptic, purified, vialed, cryopreserved formulation of Pf∆slarpb9, termed PfSPZ-GA1 
  • In 2016 we gained approval for use of PfSPZ-GA1 in humans by the Dutch Ministry of Infrastructure and the Environment (GGO IM-MV 15-004 and GGO IM-MV 15-009)
  • The ethical and medical approval to perform a clinical trial using PfSPZ-GA1 in test subjects in the Netherlands was gained in 2017 from the Central Committee on Research Involving Human Subjects (Centrale Commissie Mensgebonden Onderzoek; CCMO NL55657.000.16)
  • PfSPZ-GA1vaccine safety and efficacy trials will be conducted in human volunteers at the LUMC and RadboudMC in 2017

GAPs explained

(see also below for more detailed information and references)

What is a genetically attenuated parasite (GAP) malaria vaccine?

  • A GAP vaccine consists of live but attenuated parasites
  • GAP attenuation is achieved through genetic modification, i.e. by deleting essential genes from the genome of the malaria parasite
  • The loss of these genes from the parasite genome must ensure the complete arrest of the parasite in the liver.
  • The Leiden Malaria Research Group is at the forefront of global research focused on using live-parasites as a potential malaria vaccine. Notably, the LUMC were one of the first groups to develop the concept of immunization with GAP, showing that GAP based immunity can effectively protect mice against malaria.

How would a GAP-based vaccine work?

  • A GAP vaccine consists of live but attenuated parasites (so-called sporozoites). These sporozoites are able to invade the liver but are unable to produce the infectious ‘merozoites’ that can establish a pathogenic blood stage infection.
  • Vaccination with these sporozoites induces immune responses that protect the host from re-infection. Specifically, these immune responses kill the sporozoites that are introduced by a mosquito before they get into the liver or in the liver and therefore they prevent the pathogenic blood-stage infection.

What are the benefits of vaccination with live-parasites over subunit vaccines (which consist of only specific parasite proteins)?

  • Indeed, subunit vaccines are easier and cheaper to produce, and generally easier to store and administer than vaccines based on live parasites.
  • However, until now no subunit vaccine has been produced that induces high-level protective immunity in humans that is comparable to that achieved through immunization with live, attenuated sporozoites.
  • Immunization with live sporozoites that have either been attenuated by radiation or have been administered by mosquito bite in the presence of anti-Plasmodium chemoprophylaxis induces strong protective immunity.
  • The RUNMC malaria group in Nijmegen (The Netherlands) has demonstrated that complete and long-lived immunity in humans can be achieved with only low numbers of sporozoites (i.e. limited numbers of infected mosquito bites) administered in the presence of antimalarial prophylaxis.

Why use sporozoites that have been attenuated by genetic modification and not by other means, for example by radiation?

  • It has been shown in rodent models of malaria that GAP sporozoites can produce protective immune responses equal to, or even greater, than is produced by sporozoites that are attenuated by radition (RAS sporozoites).
  • GAP vaccines constitute a homogeneous parasite population with a distinct genetic identity, and their attenuation is not dependent upon external factors.
  • GAP can be further genetically modified to increase their immunogenicity, for example by introducing genes that encode molecules that can optimize their recognition by the immune system.

syringes

Additional (background) information

Plasmodium falciparum is the human parasite responsible for the vast majority of malaria associated morbidity and mortality; with over 200 million people infected resulting in an estimated 1 million deaths annually (WHO). Years testing a large number of (recombinant) subunit vaccines, designed to a variety of parasite antigens, have all have failed to induce sterile and long-lasting protective immunity in humans, consequently renewing an interest in vaccination with live-attenuated parasites (Luke and Hoffman, 2003; Pinzon-Charry and Good, 2008). 

Indeed, high-level (>90%) protection in humans has been achieved through immunization with live attenuated parasites; either based on immunisation with live irradiated sporozoites that developmentally arrest in the liver or via sporozoites administered by mosquito bite in the presence of anti-Plasmodium chemoprophylaxis (Luke and Hoffman, 2003; Roestenberg et al., 2009) . Indeed the RUNMC malaria group  in Nijmegen (The Netherlands) first demonstrated that immunisation with sporozoites administered under antimalarial prophylaxis can induce sustained sterile immunity in humans (Roestenberg et al., 2009; Roestenberg et al., 2011).

Recently immunisation studies with live sporozoites attenuated by genetic modification have gathered much attention as they have been shown to produce protective immune responses equal to, or even greater than, those produced by irradiated sporozoites in rodent models (Khan et al., 2012; Nganou-Makamdop and Sauerwein, 2013). These so-called genetically attenuated parasites (GAP) offer several advantages over radiation-based attenuation as they constitute a homogeneous population with a distinct genetic identity, and their attenuation is not dependent upon external factors (e.g. radiation, host drug metabolism).

The Leiden Malaria Research Group is at the forefront of international research on malaria vaccine development using live-parasites for immunisation. The LUMC were one of the first groups to develop the concept of immunization with GAP and showed that GAP based immunity can effectively protect mice against malaria (van Dijk et al., 2005). The malaria groups of the LUMC and RUNMC have translated these findings from murine malaria into the generation of a human, P. falciparum, GAP (Annoura et al., 2012). The LUMC together with RUNMC hold one of the first patents on the use of GAPs for vaccination (patent #: US7550138B1). Collectively the LUMC and RUMC are experts in Plasmodium genetic modification, immunization and protection studies in both rodents and humans malaria and have combined to generate GAP and to develop robust pre-clinical screening protocols to evaluate their suitability for vaccination.

As part of a TI-Pharma funded project RUNMC and LUMC along with their American industrial partner, SANARIA, have created a new human GAP (Pf∆slarp∆b9), where 2 genes have been removed from the parasite genome in order to ensure complete liver-stage arrest. This is the first human GAP where 2 genes (slarp and b9) governing critical, but independent, cellular process have been deleted. This human GAP and the equivalent rodent GAP has been evaluated in both preclinical safety and efficacy studies (Annoura et al., FASEB J 2014; van Schaijk et al. eLIFE 2014). Moreover, this GAP was generated using constructs that permitted the removal of the drug selectable that not only permitted the generation of multiple gene deletion mutants but also addresses safety issues concerning the presence of heterologous DNA in genetically modified organisms used in human immunization. These findings strengthen the case for further clinical development of the Pf∆slarp∆b9 GAP and its testing in humans. 

This is an important milestone for malaria research in Leiden and is the culmination of studies that were first initiated here in the mid-90s:

  • The Leiden Malaria group was the first group to develop genetic modification in malaria parasites (2 papers published in Science in 1995 and 1996)
  • Through analysis of gene-deletion mutants in rodent models of malaria, we discovered that it was possible to create attenuated parasites, which were able to invade the liver but were unable to proceed into the pathogenic blood stage infection. Importantly mice infected (immunised) with these live-attenuated parasites developed protective immunity against an infection with wild type parasites (published in PNAS in 2005).
  • In 2008, in the group of Shahid Khan, we started to work on translating these findings into a human vaccine against malaria. This was performed in collaboration with the RadboudUMC (Nijmegen) and the US company Sanaria, and was supported by a grant provided by TI-Pharma. This involved testing and refining many live genetically-attenuated vaccines until we could demonstrate that one met all the necessary pre-clinical safety and protective efficacy standards; it is now ready to advance into testing in humans.  

More details on the generation and pre-clinical characterisation of this vaccine can be found in 2 papers published in 2014; one published in the FASEB Journal in May and one that has just appeared in eLIFE.

More information about vaccination with live attenuated parasites (radiation and genetically attenuated parasites) can be found in the following papers:

  • Review on how to perform experimental malaria infections in humans, i.e. so called Controlled Human Malaria Infections (for testing vaccine efficacy in humans).
  • A N. Engl J. Med. paper showing induction of complete protective immunity in humans after only a limited number of mosquito bites.
  • A review on human immunization trails performed using radiation attenuated sporozoites administered by mosquito bite.
  • A Science paper demonstrating protective immunity in humans after immunization with radiation-attenuated and cryopreserved sporozoites administered by intravenous injection.
  • A PNAS paper (from the Leiden Malaria Research Group) was one of the first proof of concept studies demonstrating sterile immunity could be achieved using genetically attenuated sporozoites in rodent models of malaria.
  • A Vaccine paper ((from the Leiden Malaria Research Group) desribing the preclinical testing of vaccines consiting of genetically attenuated parasites
  • A review paper (from the Leiden Malaria Research Group) on the considerations behind and the progress in developing genetically attenuated sporozoites suitable for vaccination in humans
  • FASEB J and eLIFE papers describing the generation and pre-clinical characterisation of the human GAP vaccine Pf∆slarp∆b9