Localization of putative Onchocerca volvulus antigens by in
situ hybridization and immunochemistry
by Rocky S. Tuan
Our efforts during this past year have been devoted to two main areas:
1) application of the existing histological techniques of in situ
hybridization (ISH) and immunohistochemistry (IMH) to localize gene expression
of antigen of O. volvulus 3in various developmental stages of the
parasite, using cloned DNA's and antibodies directed against recombinant
antigens provided by investigators in the field; and
2) further development of ultrastructural protocols using the scanning electron
microscope (SEM) for ISH and IMH mapping of antigen expression.
Localization of Gene Expression by ISH and IMH
These studies have been carried out in collaboration with the following
investigators: Francine Perler (New England Biolabs), Thomas Unnasch (University
of Alabama), Sara Lustigman (New York Blood Center), Alan Scott (Johns Hopkins
University), A. E. Bianco (Liverpool School of Tropical Medicine), and R.
Chandrashekar (Washington University). Parasite specimens of various developmental
stages, including larval (L2 and L3, both free and embedded within the blackfly
host) and adult/microfilarial (contained within human skin nodules), have
been processed for ISH and IMH. For ISH, the DNA probe is biotin-labelled
and hybridization is detected via biotin-labelled secondary antibodies and
streptavidin-peroxidase histochemistry. The antigens (and the available
probes) include:
Perler: O13/O15 (DNA and antibodies)
Unnasch: RAL-2 (DNA and antibodies)
Lustigman: OV7 (DNA and antibodies) and OV103 (DNA)
Scott: M3/M4 (DNA)
Bianco: B20 (DNA)
Chandrashekar: OV1C (DNA), OV-1 and OV-5 (antibodies)
All of these studies have been completed, with each ISH and IMH repeated
two to three times to ensure consistent signals. All specimens are examined
by Nomarski differential interference optics and recorded using Kodak Ektar
film. The results have revealed significant differences among these antigens
in terms of tissue/cell distribution as well developmental stage specificity.
This information should be useful for the evaluation of these antigens as
potential vaccine candidates.
Ultrastructural SEM Procedure for IMH and ISH
The histolocalization procedures we have used for the gene expression studies
described above utilize sections of paraffin-embedded parasite specimens.
The fixation and embedding conditions are such that both mRNA and proteinaceous
antigens appear to retain their reactivity with the respective DNA probe
and antibodies. The calorimetric signals generated via enzyme-catalyzed
chromogenic reactions permit observations at the light microscopy level,
and with the use of Nomarski differential interference optics yield excellent
resolution of the distribution and location of the mRNA- and antigen-derived
signals. To gain further insight into the ultrastructural aspects of gene
expression, it is preferable to carry out such analysis at the electron
microscopy level. In this funding period, we have developed the following
protocol for SEM analysis of antigen expression.
The protocol utilizes specimens which have already been processed into 8
µm tissue sections placed onto a glass slide, thereby allowing
access of the DNA/antibody probes. Briefly, the procedure involves: 1) for
IMH, incubation of the immuno-reacted sections with latex-polystyrene beads
conjugated with Protein A (Polysciences, Inc.); and 2) for ISH, sequential
incubation of the sections, which have been hybridized with biotin-labelled
probe, with antibodies against biotin and Protein A-conjugated beads. Since
the Protein A-conjugated beads are also tagged with a fluorescent dye, the
efficacy of the labelling procedure is conveniently established prior to
processing for SEM (i.e. dehydration and critical point drying).
Figure 1
Results we have obtained at this point are very promising and representative
micrographs are shown in the accompanying figure (Figure 1). To summarize,
the SEM IMH analysis provides high resolution, discrete localization of
the antigen, yielding information consistent with that seen previously by
light microscopy; O13/O15 and RAL2 have been examined so far. The results
on the SEM ISH are more preliminary and will require additional troubleshooting.
The main technical difficulty here is that the extensive digestion required
to render the mRNA accessible to the probe also increases the "stickness"
of the specimen, resulting in a higher level of background binding by the
polystyrene-latex beads.
Figure 2
We are currently testing various incubation conditions to overcome this
difficulty, which we believe is surmountable. [A presentation of our recent
findings using SEM was made in the 50th Meeting of the Electron Microscopy
Society of America held in Boston, MA, in August, 1992 (Immunohistochemical
localization of gene expression in Onchocerca volvulus using latex
spheres as SEM marker by Kreitzer, Tuan and Shepley)].
SEM Morphology of Insect Stage O. volvulus L3 Larvae
An additional accompanying project which we have undertaken while processing
the various parasite specimens, is to carry out a detailed SEM study of
the L3 larvae located within the mouthpart of the Simulium fly, to
gain some insight as to how the larvae invade and inhabit the fly head.
Specimens fixed in Carnoy's solution are dehydrated and dried for SEM. The
labial structures of the fly proboscis are then sequentially and carefully
removed to uncover the internally localized L3 larvae. The observations
are shown in the accompanying figure (Figure 2). A large number of L3 larvae,
up to 15-20, are usually found located within the opening of the labia,
and are aligned along the axis from the labial pharynx to the labia, i.e.
along the axis of the fly head. One end of the larva therefore lies deep
within the mouth cavity, whereas the other end is situated close to the
mandibular opening. This type of arrangement most likely contributes to
the easy delivery of the parasite to the human host as a result of the insect
bite. How the larvae migrate from the thoracic muscle, concomitant with
an L2 to L3 molting stage, to their location in the mouthpart is presently
poorly understood. We intend to carry out additional SEM observation of
the fly at various stages after ingestion of the microfilariae to gain further
insight into this interesting developmental event in the life cycle of the
parasite.
Figure Legends
Figure 1. Localization of O13/O15 antigen in O. volvulus within human
skin nodule by SEM IMH. (A) Intrauterine, mature microfilariae (MF) are
seen decorated with 1 µm beads (arrows) in sample incubated with antibodies
to O13/O15. (B) Control treated similarly except that the primary antibodies
are omitted from the incubation mixture, showing absence of beads. C, cuticle;
H, hypodermis; M, muscle layer; U, uterine wall. Magnification bar = 10
µm.
Figure 2. SEM view of Simulium stage O.volvulus L3 larvae.
(A) Frontal low magnification view of the fly head structure, clearly displaying
the labia of the proboscis. (B) Exposure of abundant L3 larvae (arrow) after
removal of the labia. (C) High magnification of L3 larvae, showing cuticular
ridges on the surface of the parasites. Magnification bar = 100 µm
(A) or 10 µm (B and C).