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\begin{document}

\title{Evaluation of the long-term immune responses following leishmanization
using a live- Lizard Leishmania mixed with chitin}



\author[1]{Farshid Yeganeh}%
\author[1]{Mina Noroozbeygi}%
\author[1]{Nafise Keshavarzian}%
\author[1]{Sepideh Haghdoust}%
\author[1]{Mostafa Haji Molla Hoseini}%
\affil[1]{Shahid Beheshti University of Medical Sciences School of Medicine}%


\vspace{-1em}



  
  \date{May 16, 2024}


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\begin{abstract}
\textbf{Background:} Leishmanization using non-pathogenic to human
\emph{Leishmania} spp. is considered a reliable approach to immunize
subjects against \emph{Leishmania} infection. \textbf{Objectives}: Here,
we evaluated the long-term immune responses (14 weeks) after
immunization with either live- or killed-Iranian Lizard Leishmania (ILL)
mixed with chitin microparticles (CMPs) against \emph{L. major}
infection in BALB/c mice. \textbf{Methods:} In total, nine groups of
mice were included in the study. To evaluate short-term immunity, mice
were immunized with live-ILL and three weeks later were challenged with
\emph{L. major\textsuperscript{EGFP}}. To evaluate the long-term
immunity, mice were immunized with either live- or killed-ILL, and 14
weeks after immunization were challenged with \emph{L.
major\textsuperscript{EGFP}}. A group of healthy mice who received no
injection was also included in the study. Eight weeks after the
challenge with \emph{L. major\textsuperscript{EGFP}} all subjects were
sacrificed and the parasite burden (quantitative real-time PCR),
cytokines levels (IFN-\selectlanguage{greek}γ, \selectlanguage{english}IL-4, and IL-10), \emph{Leishmania}-specific
antibody concentration, and total levels of IgG1 and IgG2a were
measured. In addition, nitric oxide concentration, and arginase activity
were evaluated. \textbf{Results:} In mice that were immunized using
live-ILL+CMP, the induced proactive immune response lasted at least 14
weeks since, when they were challenged with \emph{L. major}
\textsuperscript{EGFP} at the 14 \textsuperscript{th}-week
post-immunization, no open lesion was formed during 8 weeks follow-up,
and the footpad swelling was significantly lower than controls. As well,
they showed a significant reduction in the parasite burden in
splenocytes, in comparison to the control groups including the group
that received killed-ILL+CMP. The observed protection was associated
with a higher IFN-\selectlanguage{greek}γ \selectlanguage{english}and a lower IL-10 production by splenocytes.
Additionally, the results demonstrated that arginase activity was
decreased in the ILL+CpG group compared to other groups.
\textbf{Conclusion:} The long-term response against \emph{L. major}
infection induced by Live-ILL+CMP was more competent than the response
elicited by killed-ILL+CMP to protect mice against infection with
\emph{L. major} \textsuperscript{EGFP}.%
\end{abstract}%



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