Vaccine Development Against Onchocerca volvulus :
Lessons from a Mouse Model
by David Abraham
1. Introduction
An integral component of any effort to develop a vaccine against a human
disease is an animal model for the disease. Animal models are used to study
the interaction between disease and immunological processes and for the
screening of vaccine candidates. One of the significant limitations on
studies of the immune response to O. volvulus has been the limited
host range. Experimentally, only non-human primates such as chimpanzees
and mangabey monkeys have been shown to support complete development of
the parasite. Neither of these hosts is of practical use for the initial
phases of vaccine development. It was for these reasons that efforts have
been made to develop a mouse model for the study of immunity to O. volvulus.
Another handicap with working on O. volvulus is the difficulty
in obtaining live fresh larval stages. This difficulty has been addressed
through the use of cryopreserved larvae. However, a question to be anwered
is "does the immune system recognize and respond to cryopreserved larvae
in a manner similar to fresh larvae?".
Several studies have tried unsuccessfully to infect laboratory rodents
with O. volvulus. To determine whether mice were resistant to all
stages of O. volvulus, diffusion chamber methodologies have been
employed. Diffusion chambers are 14 mm Lucite rings sealed with membranes
of various pore sizes. Larvae are inserted into the chambers and then implanted
subcutaneously. The membrane pore size prevents the larvae from leaving
while allowing host cells and serum to enter. It could therefore be determined
whether mice are susceptible to the early larval stages, even if they are
not supportive of the complete life cycle.
2. Model Development I
The first question addressed in these studies was would cryopreserved and
fresh larvae survive and develop in diffusion chambers at equivalent rates.
The second fundamental question was whether larvae would survive and develop
at equal rates in primate and rodent hosts. Third-stage larvae (L3) were
implanted in diffusion chambers in chimpanzees, mangabey monkeys, rhesus
monkeys, squirrel monkeys and inbred strains of mice, jirds and rats for
3-63 days. At different times during the experimental period, larvae were
recovered and assessed for their viability and development. Survival and
growth rates were equal regardless of whether the implanted larvae were
fresh or cryopreserved. Survival and growth rates of the implanted larvae
did not differ among the primate and rodent hosts tested, with the exception
of squirrel monkeys and rats, which were resistant to infection. Molting
from L3 to fourth-stage (L4) larvae began on day 3 and continued through
day 14 in the primates and rodents. It was therefore concluded that the
use of cryopreserved larvae in place of fresh larvae was reasonable and
that mice could be used as a model for the study of the first few weeks
of infection.
3. Development of Protective Immunity
The next question to be addressed was whether mice could be induced to
kill larvae through immune mediated processes. BALB/cBYJ mice were immunized
by subcutaneous injection of normal, irradiated, or freeze-thaw killed Onchocerca
sp. larvae. The mice received challenge infections of O. volvulus
L3 contained in diffusion chambers implanted subcutaneously. At two weeks
postinfection, the diffusion chambers were removed and larval survival was
assessed. When mice were immunized a single time with 35 krad-irradiated
or normal O. volvulus L3, there was a significant reduction in the
survival of challenge parasites. However, there was little or no reduction
in challenge worm survival when mice were immunized a single time with freeze-thaw
killed O. volvulus L3 or L4, or irradiated O. lienalis L3.
When a second dose of freeze-thaw killed O. volvulus L3 or irradiated
O. lienalis L3 was administered, there was a significant reduction
in parasite survival in immunized mice. Immunization with O. volvulus
L4 or a combination of L3 and L4 failed to confer protection. These
results demonstrated that mice could be immunized against larval O. volvulus
and that diffusion chambers were an efficient method for studying protective
immunity to this parasite in a mouse model.
4. Mechanism of Immune Mediated Killing of Larvae
Using this mouse model the mechanism of immune mediated killing of challenge
larvae as induced by irradiated L3 was studied. Direct contact between
host cells and parasites was required for killing of larvae in immunized
hosts. To define the mechanism of immune-mediated killing in this system,
the time of influx of cells and cytokines into the infection site were compared
to the time challenge infections were killed. The only cell type that was
found to increase in diffusion chambers in immunized mice was the eosinophil;
maximal levels of eosinophils were coincident with the time of parasite
killing. IL-5 was found in diffusion chambers of immunized, but not control,
mice coincident with the time of parasite killing. Significant levels of
Interferon-g were absent in the diffusion chambers of both groups. Immunized
mice were next treated with mAb to eliminate IL-5 or IL-4 to assess the
role that these cytokines or their byproducts play in larval killing. Elimination
of either IL-5 or IL-4 significantly reduced the protective effects of vaccination
against larval O. volvulus. These findings suggests that the protective
immune response in mice to larval O. volvulus is dependent on eosinophils
(as shown by IL-5 dependency) and IgE or IgG1 (as shown by IL-4 dependency).
Humoral responses of immunized mice to O. volvulus L3 antigens
were also examined. ELISA measurements of total serum antibody levels indicated
that IgE was the only antibody isotype elevated in mice immunized with O.
volvulus L3. IgM from immunized mice was the only isotype that recognized
surface antigens on intact O. volvulus L3. IgG1, IgG3, IgE and
IgA recognized internal parasite antigens on O. volvulus L3 frozen
sections. Western blot analysis of L3 proteins showed that the serum from
mice immunized with O. volvulus L3 IgG1, IgG2a/2b, IgA, and IgE,
as well as IgM, recognized unique L3 proteins. Antibodies in serum from
L3 immunized mice were able to detect O. volvulus adult antigens
in a pattern similar to the recognition found in O. volvulus L3.
Some L3 antigens were shared by adults, while other antigens were L3 specific.
The ELISA, immunohistochemistry and Western blot findings thus demonstrated
a complex pattern of parasite antigen recognition by antibodies found in
mice immune to the L3 of O. volvulus.
5. Summary of Antigen Screen
The mouse model was also used to screen recombinant antigens supplied by
collaborators, as arranged by the Onchocerciasis Task Force of the Edna
McConnell Clark Foundation. A variety of criteria were used in the selection
of these antigens, all of which indicated that a particular antigen might
have vaccine potential. The following immunization protocol was used.
Mice were injected subcutaneously with 25 mg of a particular antigen mixed
with a water-in-oil block copolymer adjuvant. This was followed four weeks
later with an identical booster immunization; challenge infections were
given two weeks later in diffusion chambers which were implanted for two
weeks. Six out of the 16 antigens tested induced protective immunity in
single experiments using challenge L3 which were prepared in Liberia.
When these experiments were repeated with L3 from Cameroon protective immunity
was not observed with any of the antigens. Results from all of the single
antigen immunizations are summarized in the accompanying table.
6. Model Development II
As is clear from the table, significant differences were seen in the ability
of mice to kill L3 dependent on the source of the larvae. The reason for
the change in the source of the larvae was due to political unrest in Liberia.
The phenomenon of resistance to immune mediated killing of larvae prepared
in Cameroon was also seen in mice immunized with irradiated larvae. This
finding precipitated a series of experiments whose goal it was to determine
why protective immunity was no longer evident in the mouse model.
Immune mediated killing of challenge larvae was not observed using proven
protocols with irradiated larvae, dead larvae, or recombinant antigens nor
with new immunization methods using different routes and various adjuvants.
It was determined that there was no change in the mouse strain, the x-ray
dose, or the materials making up the diffusion chambers. At this point
it was concluded that something had changed with the larvae and not with
the experimental procedure; it was not known, however, whether the problem
in demonstrating protective immunity was in the inability of the larvae
to induce protective immunity, or if the failure was because the larvae
were resistant to the induced immunity.
It was hypothesized, based on observation of larval-specific immune responses
in mice immunized with the cryopreserved larvae, that the deficit in the
system was in the ability of the larvae to be killed. It was further hypothesized
that the cryopreservation process itself was affecting larval susceptibility
to immune-mediated killing. To test these hypotheses, mice were immunized
with cryopreserved L3 from Cameroon. Each immunized and control animal
received two chambers with challenge larvae. One chamber contained fresh
larvae (i.e. not cryopreserved) and the other chamber contained cryopreserved
L3 from Cameroon. Diffusion chambers were implanted for 14 days after which
they were recovered. The results from this experiment showed that there
was a 50% reduction in fresh larval survival in immunized mice whereas cryopreserved
worms were not killed. These results were especially compelling because
fresh and cryopreserved larvae were implanted in the same animals.
The previous experiment conclusively demonstrated that immunization with
cryopreserved L3 from Cameroon was effective at inducing protective immunity
to the larval stage. Previous studies using larvae prepared in Liberia,
showed that the challenge larvae were killed by day 5 post challenge. All
of the experiments described above had diffusion chambers implanted for
14 days, thus allowing almost three times the required amount of time for
killing to occur. It was next hypothesized that larvae cryopreserved in
Cameroon might require more time to be killed and that they would be killed
if they were implanted in vivo for more than two weeks. In the
experiment designed to test this hypothesis it was determined that killing
of cryopreserved challenge larvae was seen at three and at four weeks post
challenge. It was also noted that larval development from L3 to L4 was
retarded in immunized mice at the two week time point. Larval developmental
retardation in immunized animals has been observed with several other filarial
parasites. Therefore, immunity against the cryopreserved L3 from Cameroon
was demonstrated by developmental retardation at two weeks and by larval
death beginning at three weeks post-challenge.
To further test the effect of cryopreservation on the ability of larvae
to induce or be killed by the immune response, the following experiment
wasperformed. Mice were immunized with larvae prepared in Cameroon which
were cryopreserved using different methods. The mice then received challenge
infections consisting of either homologously or heterologously
cryopreserved larvae. In these experiments it was concluded that the cryopreservation
method currently in use in Cameroon prepared the larvae such that immunization
with them lead to immunity to larvae prepared with all other cryopreservation
methods. The reverse was, however, not true; other methods of larval cryopreservation
did not result in larvae which
were capable of inducing protective immunity. A cryopreservation method
has therefore been identified that will yield larvae which are capable of
inducing immunity to fresh larvae and to larvae cryopreserved using a variety
of methods. These cryopreserved larvae thus provide the closest representation
of that which occurs with fresh larvae.
7. Conclusion
Progress towards developing a vaccine against onchocerciasis has been diverted
during the past months because of a failure in the mouse model to demonstrate
protective immunity. This problem has been solved and it has been demonstrated
that the lack of protective immunity was caused by a change in the manner
in which the L3 were being cryopreserved. This diversion has brought to
light an issue of critical importance which would have otherwise been overlooked.
Experiments have been concluded in which the appropriate method for larval
cryopreservation has been determined. The final result is an animal model
which will mimic human disease more accurately and thus be of value as a
tool in the effort to develop a vaccine against onchocerciasis.
Summary of recombinant antigens in the mouse/diffusion chamber screen
(Abraham)
| Antigen |
Source |
% Reduction in Larval Survival |
Source of L3 |
| Oncho C-27 |
McKerrow |
0 |
Liberia |
| OI-3 |
Perler |
35* |
Liberia |
| OI-3 |
tString |
0 |
Cameroon |
| OV-7 |
Lustigman |
33* |
Liberia |
| OV-7 |
tString |
0 |
Cameroon |
| Paramyosin |
McReynolds |
0 |
Liberia |
| RAL-2 |
Unnasch/Chakravarti |
45* |
Liberia |
| RAL-2 |
tString |
0 |
Cameroon |
| OV-9 |
Lustigman |
42* |
Liberia |
| OV-9 |
tString |
0 |
Cameroon |
| OVSOD |
Henkle |
0 |
Liberia |
| OVL3 |
Lucius |
0 |
Liberia |
| OV-11 |
Bradley |
42* |
Liberia |
| OV-11 |
tString |
0 |
Cameroon |
| MOV-14 |
Bianco |
0 |
Liberia |
| Oncho-1 |
McKerrow |
0 |
Liberia |
| B-20 |
Bianco |
39* |
Liberia |
| B-20 |
tString |
0 |
Cameroon |
| RAL-6 |
Unnasch |
0 |
Cameroon |
| OVGST |
Henkle |
0 |
Cameroon |
| RAL-1 |
Unnasch |
0 |
Cameroon |
| EGP GG316 |
Unnasch |
0 |
Cameroon |