Biology Reference
In-Depth Information
Chapter 8
tebenquichense
Total Polar Lipids
Raul O. Gonzalez, Leticia H. Higa, Romina A. Cutrullis, Marcos Bilen,
Irma Morelli, Diana I. Roncaglia, Ricardo S. Corral, Maria Jose Morilla,
Patricia B. Petray, and Eder L. Romero
INTRODUCTION
Archaeosomes (ARC), vesicles prepared from total polar lipids (TPL) extracted from
selected genera and species from the Archaea domain, elicit both antibody and cell-
mediated immunity to the entrapped antigen, as well as efficient cross priming of
exogenous antigens, evoking a profound memory response. Screening for unexplored
Archaea genus as new sources of adjuvancy, here we report the presence of two new
Halorubrum tebenquichense
strains isolated from gray crystals (
GCs
) and black mud
(
BM
) strata from a littoral Argentinean Patagonia salt flat. Cytotoxicity, intracellular
transit, and immune response induced by two subcutaneous (sc) administrations (days
0 and 21) with bovine serum albumin (BSA) entrapped in ARC made of TPL either
form
BM
(ARC-
BM
) and from
GC
(ARC-
GC
) at 2% w/w (BSA/lipids), to C3H/HeN
mice (25 μg BSA, 1.3 mg of archaeal lipids per mouse) and boosted on day 180 with
25 μg of bare BSA, were determined.
The DNA G + C content (59.5 and 61.7% M
BM
and
GC
, respectively), 16S rDNA
sequentiation, DNA-DNA hybridization, arbitrarily primed fi ngerprint assay, and bio-
chemical data confi rmed that
BM
and
GC
isolates were two non-previously described
strains of
H. tebenquichense
. Both multilamellar ARC mean size were 564 ± 22 nm,
with -50 mV zeta-potential, and were not cytotoxic on Vero cells up to 1 mg/ml and up
to 0.1 mg/ml of lipids on J-774 macrophages (XTT method). The ARC inner aqueous
content remained inside the phago-lysosomal system of J-774 cells beyond the fi rst
incubation hour at 37°C, as revealed by pyranine loaded in ARC. Upon sc immuniza-
tion of C3H/HeN mice, BSA entrapped in ARC-
BM
or ARC-
GC
elicited a strong and
sustained primary antibody response, as well as improved specifi c humoral immunity
after boosting with the bare antigen. Both IgG1 and IgG2a enhanced antibody titers
could be demonstrated in long-term (200 days) recall suggesting induction of a mixed
Th1/Th2 response.
We herein report the fi nding of new
H. tebenquichense
non alkaliphilic strains in
Argentinean Patagonia together with the adjuvant properties of ARC after sc admin-
istration in mice. Our results indicate that ARC prepared with TPL from these two
strains could be successfully used as vaccine delivery vehicles.
In 1997, the pioneering work of Sprott showed that parenteral administration of
nano-sized vesicles (ARC) prepared with TPL extracted from microorganisms of the
