The morbidity and mortality affiliated with vector-borne diseases are staggering, and the associated economic and social hardships are overwhelming, especially in populations without the political or financial means for effective control or treatment. Many of these diseases continue to re-emerge in former endemic areas and/or emerge in new parts of the world, and conventional means of control are often inadequate due to the appearance of pesticide-resistant vectors, drug resistant-pathogens, and the collapse of vector control programs, among other factors. At present, safe and efficacious vaccines and therapeutics for prevention and treatment of many of these diseases are lacking.
La Crosse virus (LACV), family Bunyaviridae, is a mosquito-borne pathogen and is the leading cause of pediatric arboviral encephalitis in the United States. LACV is not only an important human pathogen, but it is also a model for development of vaccines and antiviral therapies for other pathogens in the family Bunyaviridae. Many bunyaviruses are designated as National Institute of Allergy and Infectious Diseases (NIAID) priority pathogens and there is cause for concern that they could be exploited as bioweapons. Aerosol infection animal models are essential to test the efficacy of candidate vaccines and antiviral therapies for these important pathogens. There are no human vaccines or antiviral treatments for LACV or other members of the family. Accordingly, in Chapter 2, aerosol and intranasal inhalational challenge models of LACV infection in mice were developed and tested. Following aerosol or intranasal challenge with LACV, 100% of normally-resistant adult mice developed clinical signs of LAC encephalitis and died. LACV was detected in high titers in the nasal turbinates, brains and lungs of aerosol- or intranasally-challenged mice, as well as in the sera and livers of mice challenged intranasally. Brains of LACV-challenged mice exhibited histologic lesions of meningoencephalitis, and LACV RNA was detected and amplified from brains of challenged mice. To our knowledge, this is the first report of aerosol-transmission of LACV leading to the development of lethal encephalitis in adult mice. The experimental challenge models described herein should be useful tools in the eventual development of sorely-needed vaccines and antivirals for the prevention or treatment of bunyavirus aerosol infections.
Immunotherapy using cationic liposome-DNA complexes (CLDC) has been shown to promote antiviral, antitumor, and antibacterial immune responses in various experimental animal models. This protection relies on non-pathogen-specific activation of soluble and cellular innate immune effectors. In Chapter 3, we evaluated the ability of CLDC to protect adult mice from the development of encephalitis in a LACV aerosol challenge model. Both pre-challenge (prophylactic) and post-challenge (therapeutic) administration of CLDC significantly increased survival in LACV-challenged animals in this model system. Intraperitoneal administration of CLDC elicited reductions in viral titer in both peripheral tissues and the central nervous system (CNS) and decreased the severity of CNS lesions in treated mice compared to sham-treated control mice. Protection was associated with increased expression of IFN-α5 and IFN-β1 in the spleen, as well as IFN-γ in the brain. Systemic depletion of natural killer (NK) cells prior to treatment was found to abrogate the full protective ability of CLDC administration, suggesting an integral role for this cell type in CLDC-induced protection. These data indicate that CLDC is an effective antiviral immunotherapy that provides both prophylactic and therapeutic protection against an otherwise lethal aerosol challenge with a viral pathogen.
Leishmania major, one causative agent of Old World cutaneous leishmaniasis, is a vector-borne protozoan parasite transmitted by the bite of infected female phlebotomine sand flies. At present, there is no vaccine approved for use in humans for the prevention of L. major infection. Chapter 4 describes significant protection against infection with L. major induced by CLDC-based immunization against the immunomodulatory salivary peptide maxadilan (MAX) from the sand fly Lutzomyia longipalpis. Following intraperitoneal or subcutaneous immunization with MAX, both lesion sizes and parasite burdens of infected footpads of immunized mice were significantly reduced in comparison to those of non-immunized mice. The protection elicited using a CLDC adjuvant exceeded that elicited using a human-approved aluminum hydroxide adjuvant (Alhydrogel®); additionally, notable inflammation and tissue damage present at the site of immunization when Alhydrogel® was employed as adjuvant was completely absent in mice immunized with CLDC as adjuvant. Intracellular cytokine staining of CD4+ lymphocytes identified important differences in IFN-γ and IL-4 production in the context of infection due to immunization or lack thereof. The protection described herein highlights the importance of salivary immunomodulation in the initiation of vector-borne pathogen infections, and provides compelling evidence in support of the inclusion of salivary molecules as antigens in the formulation of subunit vaccines intended to protect against transmission of arthropod-borne pathogens.
In these studies, we utilized CLDCs as both immunotherapeutics and vaccine adjuvants to induce innate and Th1-type immune responses protective against infection with LACV and L. major, both of which are arthropod-borne intracellular pathogens that require the induction of pro-inflammatory Th1-biased immune responses for clearance. This body of work presents evidence that CLDC administration induces protective immune responses against aerosolized LACV challenge and parenteral challenge with L. major, suggesting that CLDCs are versatile and effective vehicles for the elicitation of immune protection against pathogens susceptible to pro-inflammatory and Th1-biased immune responses, and are worthy of future exploration and further application.