short-term immunity and limited cross-protection against heterologous serovars of pathogenic leptospires [4, 5].
With the availability of whole-genome sequences from multiple
strains of Leptospira [6–8], current vaccine research has focused on
identifying conserved leptospiral proteins, which have the potential
of eliciting cross-protective immunity. Surface-exposed outer membrane proteins that are recognized by the host immune response
early during the infection and play a role in bacterial virulence are
ideal targets for vaccine development.
Vaccine studies require the use of animals. Hamsters are one of
the most common animal models of leptospirosis because they are
highly susceptible to leptospiral infection and exhibit clinical symptoms that mimic severe human leptospirosis [9]. Animal experiments necessitate the use of low in vitro passage, highly virulent
strains of Leptospira spp., and the preliminary determination of the
median endpoint dose (ED 50 ), i.e. the number of bacteria with
which 50% of animals meet the endpoint criteria. The ED 50
depends on the serovar and strain used, the number of in vitro
passages, and the animal model. A valid challenge requires that
more than 80% of the unvaccinated control animals succumb to
lethal infection [10], but the goal is that all animals in the control
group reach the endpoint criteria.
Here we describe how a leptospiral protein is tested as vaccine
candidate using golden Syrian hamsters. The coding sequences of
the protein lacking the signal peptide are amplified from the Leptospira genome, cloned, and expressed as a recombinant protein,
often with a small tag used for affinity purification of the protein.
The purified protein (antigen) is mixed with an adjuvant and
injected into hamsters. Animal serum is collected prior to and
during the immunization protocol to measure the humoral
immune response generated by the candidate protein using
enzyme-linked immunosorbent assay (ELISA). Kidney colonization in survivors is assessed by in vitro culture in EMJH medium
and quantitative PCR. An ideal subunit vaccine must have the
ability to protect hamsters from lethal infection and provide sterilizing immunity.
Using these methods, we were able to show that hamsters
immunized with a C-terminal fragment (domains 7–13) of leptospiral immunoglobulin (Ig)-like protein A (LigA), an outer membrane lipoprotein, survived intraperitoneal bacterial challenge with
L. interrogans (Fig. 1) and generated a humoral immune response
against LigA (Fig. 2). However, LigA failed to protect against
kidney colonization by L. interrogans [11].
258
Karen V. Evangelista and Kristel Lourdault
With the availability of whole-genome sequences from multiple
strains of Leptospira [6–8], current vaccine research has focused on
identifying conserved leptospiral proteins, which have the potential
of eliciting cross-protective immunity. Surface-exposed outer membrane proteins that are recognized by the host immune response
early during the infection and play a role in bacterial virulence are
ideal targets for vaccine development.
Vaccine studies require the use of animals. Hamsters are one of
the most common animal models of leptospirosis because they are
highly susceptible to leptospiral infection and exhibit clinical symptoms that mimic severe human leptospirosis [9]. Animal experiments necessitate the use of low in vitro passage, highly virulent
strains of Leptospira spp., and the preliminary determination of the
median endpoint dose (ED 50 ), i.e. the number of bacteria with
which 50% of animals meet the endpoint criteria. The ED 50
depends on the serovar and strain used, the number of in vitro
passages, and the animal model. A valid challenge requires that
more than 80% of the unvaccinated control animals succumb to
lethal infection [10], but the goal is that all animals in the control
group reach the endpoint criteria.
Here we describe how a leptospiral protein is tested as vaccine
candidate using golden Syrian hamsters. The coding sequences of
the protein lacking the signal peptide are amplified from the Leptospira genome, cloned, and expressed as a recombinant protein,
often with a small tag used for affinity purification of the protein.
The purified protein (antigen) is mixed with an adjuvant and
injected into hamsters. Animal serum is collected prior to and
during the immunization protocol to measure the humoral
immune response generated by the candidate protein using
enzyme-linked immunosorbent assay (ELISA). Kidney colonization in survivors is assessed by in vitro culture in EMJH medium
and quantitative PCR. An ideal subunit vaccine must have the
ability to protect hamsters from lethal infection and provide sterilizing immunity.
Using these methods, we were able to show that hamsters
immunized with a C-terminal fragment (domains 7–13) of leptospiral immunoglobulin (Ig)-like protein A (LigA), an outer membrane lipoprotein, survived intraperitoneal bacterial challenge with
L. interrogans (Fig. 1) and generated a humoral immune response
against LigA (Fig. 2). However, LigA failed to protect against
kidney colonization by L. interrogans [11].
258
Karen V. Evangelista and Kristel Lourdault