Abstract
Leishmaniasis is a neglected protozoan disease affecting over 12 million people globally with no approved vaccines for human use. New World cutaneous leishmaniasis (CL) caused by L. mexicana is characterized by the development of chronic non-healing skin lesions. Using the CRISPR/Cas9 technique, we have generated live attenuated centrin knockout L. mexicana (LmexCen−/−) parasites. Centrin is a cytoskeletal protein important for cellular division in eukaryotes and, in Leishmania, is required only for intracellular amastigote replication. We have investigated the safety and immunogenicity characteristics of LmexCen−/− parasites by evaluating their survival and the cytokine production in bone-marrow-derived macrophages (BMDMs) and dendritic cells (BMDCs) in vitro. Our data shows that LmexCen−/− amastigotes present a growth defect, which results in significantly lower parasitic burdens and increased protective cytokine production in infected BMDMs and BMDCs, compared to the wild type (WT) parasites. We have also determined the safety and efficacy of LmexCen−/− in vivo using experimental murine models of L. mexicana. We demonstrate that LmexCen−/− parasites are safe and do not cause lesions in susceptible mouse models. Immunization with LmexCen−/− is also efficacious against challenge with WT L. mexicana parasites in genetically different BALB/c and C57BL/6 mouse models. Vaccinated mice did not develop cutaneous lesions, displayed protective immunity, and showed significantly lower parasitic burdens at the infection site and draining lymph nodes compared to the control group. Overall, we demonstrate that LmexCen−/− parasites are safe and efficacious against New World cutaneous leishmaniasis in pre-clinical models.
Highlights
Leishmania (L.) mexicana is the most prevalent causative agent of cutaneous leishmaniasis (CL) in North and Central America[1,2].L. mexicana causes extensive skin lesions that, in some cases, further develop into diffuse sores on different parts of the body and can spread to the mucosa[1,2]
Wildtype (WT) L. mexicana promastigotes were transfected with the pLdCN a&b CRISPR vector (Fig. 1a) containing a neomycin (G418)-resistant gene, and expressing the Cas[9] nuclease and the two guide RNAs a and b, targeting the 5′ and 3′ flanking sequences of the centrin gene (LmxM.22.1410) (Fig. 1b)
Inducing protection against L. mexicana challenge will more likely be achieved with a homologous vaccine, and it is important to develop a vaccine strain specific for American leishmaniasis
Summary
Leishmania (L.) mexicana is the most prevalent causative agent of cutaneous leishmaniasis (CL) in North and Central America[1,2].L. mexicana causes extensive skin lesions that, in some cases, further develop into diffuse sores on different parts of the body and can spread to the mucosa[1,2]. Leishmania (L.) mexicana is the most prevalent causative agent of cutaneous leishmaniasis (CL) in North and Central America[1,2]. CL lesions can ulcerate and give rise to disfiguring scars, leading to long-lasting disabilities, social stigma, poverty, and psycho-sociological trauma[3–6]. These issues impact an individual’s health and quality of life and result in an overall economic strain, especially for low- and middle-income countries (LMIC)[3,7,8]. A protective immune response against CL is characterized by the activation of phagocytes, such as macrophages and dendritic cells (DCs), and by the subsequent induction of Th1-polarized responses[12]. Interleukin (IL)-12, secreted by phagocytes, promotes differentiation of Th0 into Th1 cells and subsequent release of interferon-γ (IFN-γ), crucial for combating leishmaniasis[12]. While a Th1 immune response is mostly protective, a Th2 response, characterized by IL-10 and IL-4, confers susceptibility to CL12–15
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