Introduction and Background of the Project

Introduction

Human infection by the parasite Trypanosoma cruzi causes Chagas disease which is the leading infectious cause of heart failure in Latin America. Approximately 20-30% of those chronically infected develops cardiac fibrosis and associated cardiomyopathy. Chagas disease is increasingly found outside of Latin America mainly due to trans-migration of chronically infected individuals and affects at least 300,000 people in the United States and 8-11 million worldwide. There are increasing numbers of people infected by T. cruzi in the lower 20 of the United States due to exposure to infected insect vectors. Identification and treatment of infected people are challenging and only two rather antiquated drugs (nifurtimox and benznidazole) are available to treat the infection but are ineffective to completely clear the parasites from the host and have significant side effects which hamper their use. Thus there is an urgent need to develop a safe vaccine for prevention of T. cruzi infection and to develop more therapeutic strategies for treatment. Our laboratory is currently characterizing the biochemical and biological properties of T. cruzi cyclophilin 19 (Cyp19), a peptidyl-prolyl-isomerase that catalyzes the cis-trans isomerization of various cellular proteins acting as a chaperone. We created a double allelic knock-out parasite (DKO) line devoid of Cyp19 expression, which has shown T. cruzi Cyp19 as an indispensable protein for parasite infectivity and virulence supporting the hypothesis that this protein represents a potential critical target for small molecule inhibitors to treat the infection. Although unable to cause disease in animals, repeated immunization with live DKO parasites stimulates anti-parasitic immunity which is completely protective to mice in a model of acute Chagas disease, demonstrating proof-of-concept that this is a promising live attenuated vaccine strain.

Project objective

The long-term goal of this proposal is to generate a safe and highly efficacious live attenuated vaccine for Chagas disease for use in humans and in animals. The specific objectives are: 1) engineering a Cyp19 DKO (CC-DKO) vaccine strain using CRISPR/Cas9 and confirming its potential to provide protection in mice; 2) understanding of the T- and B-cell response responsible for protective immunity generated by this vaccine strain; 3) demonstrating that the CC-DKO vaccine is efficacious at preventing acute and chronic Chagas disease in mice and cross-protects from multiple strains and understand the duration of immunity; 4) understanding if the vaccine is safe in immunosuppressed hosts; and 5) understand if the CC-DKO is transmissible through the insect vector.

Project design

The overall design of the project is divided into the following specific aims:

Aim 1: Engineer and test a Cyp19 DKO line using CRISPR/Cas9 technology and perform characterization, stability, safety, and toxicity of this version of the vaccine.

Aim 2: Test CC-DKO-strain vaccinated animals for the determinants of protective immunity, to understand what profile of T- and B-cell immunity is provoked by vaccination and to optimize the vaccine strategy.

Aim 3: Determine if DKO vaccination leads to protection of chronic Chagas disease in the mouse model of infection, test the duration of immune-protection and the ability of the vaccine to cross-protect from diverse strains of T. cruzi.

Aim 4: Test the ability of the DKO strain to grow, survive in triatomine insects and be transmitted between animals and insects.

How can your partnership (project) address global health challenges?

The results of this work will lead to the development of a vaccine against Chagas disease which affects millions of people living in endemic areas in Central and South America. This infection is the leading cause of heart failure in these regions, thus this vaccine will help decrease the morbidity and mortality associated with this infection.

What sort of innovation are you bringing in your project?

The innovation in this project related to the development and testing of a novel live attenuated vaccine strain using CRISPR/Cas technology for an infectious parasitic disease in which there are currently no licensed prophylactic vaccines.

Role and Responsibility of Each Partner

Dr. McGwire (Ohio State University) will manage the project, obtain regulatory clearances, ensure regulatory compliances, distribute of budget, and progress reporting to GHIT.  His lab will be involved in creating the CRISPR/Cas9 version of the DKO and performing characterization, stability, and toxicity of this version of the vaccine.  They are also responsible to test the DKO vaccine strain in acute and chronic Chagas disease models and to do the challenge and cross-protection experiments. His and Dr. Satoskar’s lab will work together on analysis of the immune response of vaccinated animals.

Dr. Satoskar (Ohio State University) will help co-manage the project with Dr. McGwire and his lab will perform immune response analysis of vaccinated animals and help in infection experiments.

Dr. Hamano (Nagasaki University) will be involved in undertaking obtaining clearances for importing vaccine and WT parasite strains to Japan for pre-clinical testing at of the strains in immunosuppressed animals. He will oversee/coordinate pre-clinical testing.

Dr. Grijalva (Ohio University and CIDR, Ecuador) will obtain clearances for importing vaccine and WT parasite strains to Ecuador and coordinate the work at the CIDR will be involved in testing the ability of the DKO vaccine strain to propagate in triatomine bugs.

Dr. Villacis (CIDR, Ecuador) will help co-manage the entomological studies at the CIDR and will responsible for conducting and supervising the parasite-insect studies.

Final Report

1. Project objective:

Chagas disease is caused by the parasite Trypanosoma cruzi, which is endemic in Mexico and Central and South Americas where it is the leading cause of heart failure. There are no approved vaccines for prevention of this infection. We have previously shown that immunization with our prototype vaccine strain can protect mice from establishing infection. The objective of this project was to genetically revise and test these for protective efficacy against lethal challenge in the mouse model, safety with administered to immunocompromised mice and to analyze their growth and transmissibility by triatomine insect vectors.

2. Project design:

 Revision of our prototype vaccine strain using CRISPR/Cas modification technology was completed to inactivate antibiotic resistance markers and to further remove a cryptic copy of the cyclophilin gene. Once obtained, these mutants were used for: 1) preclinical testing in vaccination-protection studies in mice, 2) safety testing in various immunosuppressed mouse strains, and 3) testing for their growth and development in laboratory infected triatomine insects and testing for insect-to-animal and animal-to-insect transmissibility.

3. Results, lessons learned:

 The results and conclusions in the 4 main sub-project areas:

a)   Genetic modifications: We utilized the CRISPR/Cas system for in activation the antibiotic resistance genes used for the initial removal of genes for the virulence factor, cyclophilin, in the prototype strain. A second modification was made by inactivating the last cyclophilin gene in this strain. These modifications were important since the potential for utilization of this strain in higher animals would be precluded by the presence antibiotic resistance genes, and risk of reversion of the mutations in cyclophilin genes.

b)      Host infection and vaccine efficacy: When inoculated into mice, our revised vaccine strains were unable to parasitize host tissue or blood and did not cause clinical disease or impact survival. Pre-immunized with these strains were challenged with wildtype parasites were protected from developing growth of parasites in the blood and tissue and were did not develop clinical disease or have diminished survival. Non-immunized control animals developed rising parasitemia, developed parasites in tissues and had diminished survival.

c)       Safety in immunosuppressed mice: When administered to genetically engineered immunosuppressed mice, the vaccine strains were unable to replicate in or cause clinical disease. Survival of inoculated mice was the same has un-infected controls. Tissues harvested from mice inoculated with these strains did not contain viable parasites in culture assays. Immune-competent mice inoculated with the vaccine strains were treated with the immune-suppressant dexamethasone, which did not impact the survival of mice or the development of parasites in the blood or tissues.

d)      Growth and transmissibility by triatome insects: The revised mutant strains were used to infect triatomine insects (which are the insect vector responsible for the spread of T. cruzi from animals to humans). Insects fed on blood containing the vaccine strains did not develop infective stage parasites in the intestinal tract or in the feces. In order to test transmissibility, feces from these animals were administered to non-infected mice, which did not develop parasitemia or clinical disease. Insects fed on mice infected with vaccine strains did not become infected with the vaccine strains.

Overall, these results show that the revised vaccine strain is non-pathogenic in immune-competent and immune-compromised hosts, efficacious for preventing acute Chagas disease in the mouse model and safe in immunosuppressed hosts. The insect studies indicate that there is no potential for transmission of these strains by triatomine insects. Further testing for prevention of chronic disease is ongoing.