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Memórias do Instituto Oswaldo Cruz
Fundação Oswaldo Cruz, Fiocruz
ISSN: 1678-8060 EISSN: 1678-8060
Vol. 90, Num. 4, 1995, pp. 553-556
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Mem Inst Oswaldo Cruz, Rio de Janeiro, Vol.
90(4): 553-556 Jul/Aug. 1995
Meetings on Vaccine Studies towards the Control of
Leishmaniasis
UNDP/World Bank/WHO Special Program for Research and Training
in Tropical Diseases (TDR)
February 13-22 and April 4-5, 1995
Gabriel Grimaldi Jr
Departamento de Imunologia, Instituto Oswaldo Cruz, Av. Brasil
4365, 21045-900 Rio de Janeiro, RJ, Brasil
Code Number: OC95110
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Leishmaniasis is one of the major parasitic diseases targeted
by the World Health Organization (WHO 1990 Tech Rep Ser 793).
Recent epidemiologic studies indicate that the disease in the
Americas is far more abundant and of greater public health
importance than was previously recognized (G Grimaldi Jr et
al. 1989 Am J Trop Med Hyg 41: 687). Control of
leishmaniasis in this region is complicated by a variety of
leishmanial parasites (at least 13 distinct New World
Leishmania species are recognized as causing human illness)
and by the fact that each of these parasites has a unique
epidemiologic pattern (reviewed in G Grimaldi Jr, RB Tesh 1993
Clin Microbiol Rev 6: 230). These data explain the limited
success of current control strategies (based on conventional
measures such as vector reduction, elimination of infected
reservoirs, personal protection, surveillance, and treatment)
for American leishmaniases. One obvious solution to the
problem is the development of safe and effective vaccines
against the disease. However, considering the genetic
polymorphism and biological diversity of the parasites,
development of effective vaccines may be a formidable task.
Clinical and experimental evidence indicate that vector,
parasite, and host factors all influence the evolution and
outcome of leishmanial infection. A number of Leishmania
species are capable of producing a broad spectrum of disease
in humans, ranging from asymptomatic infection to horribly
disfiguring forms of mucosal leishmaniasis (ML) or the
potentially fatal visceral leishmaniasis (VL). The more benign
self-healing forms of leishmaniasis in humans usually result
in protection against reinfection, and cell-mediated immunity,
rather than humoral immunity, is considered of primary
importance in acquired resistance. Immune protection to
infection with L. major in resistant mice is associated with
selective activation and differentiation of effective CD4+
helper T (Th) cells, the Th1 subset, which are characterized
by a distinct cytokine secretion pattern (e.g., interleukine
2, IL-2; gamma interferon, IFN-g; and lymphotoxin). In
contrast, the progressive leishmanial infection in susceptible
mouse strains is correlated with activation of the Th2 CD4+
cell response, which expresses IL-4, IL-5, IL-6, and IL-10
among other cytokines (RM Locksley et al. 1991 Res
Immunol 142: 28). Several factors may affect the preferential
induction and/or expansion of distinct Th cell subsets during
leishmanial infection, including the cytokines (e.g., IL-12;
IFN-g) present during the initial events of cell
differentiation, and the interaction with regional
antigen-presenting cells (APC), which can preferentially
present different classes of antigens (P Scott 1991 J Immunol
147: 3149, FP Heinzel 1994 Parasitol Today 10: 190).
Production of IFN-g by protective T cells has also been
correlated with the ability of vaccinated and naive mice to
control infection with L. donovani (KE Squires et al.
1989 J Immunol 143: 4244). However, the differential
production of Th1- and Th2-derived cytokines does not seem to
determine the genetically controlled or vaccine-induced rate
of cure in murine visceral leishmaniasis (PM Kaye et
al. 1991 J Immunol 146: 2763). In addition, contributions
by CD8+ T cell to the mediation of protective immunity against
leishmanial infections have been shown (JO Hill et al.
1989 J Exp Med 169: 1819).
Although an interplay exist between the host immune system
and parasite pathogenic capabilities in the clinical
expression of human leishmaniasis, the mechanisms have yet to
be determined. T cell responses correlate with recovery from
and resistance to human leishmaniasis. A lymphocytic response
to leishmanial antigens usually develops during both CL and ML
but is absent in diffuse cutaneous leishmaniasis (DCL) and VL.
Conversely, anti-Leishmania antibody titers are generally low
in the sera of patients with CL or ML but moderate to high in
patients with DCL or VL (reviewed in Grimaldi, Tesh loc.
cit.). When tested in vitro, peripheral lymphocytes from
patients with CL or ML produced high levels of IFN-g in
response to parasite antigen (EM Carvalho et al. 1985 J
Immunol 135: 4144, SC Mendonca et al. 1986 Clin Exp
Immunol 64: 269, M Castes et al. 1988 J Clin Microbiol
26: 1203, S Frankenburg et al. 1993 Am J Trop Med Hyg
48: 512). There appears to be a mixed cytokine profile
associated with active CL or ML (C Pirmez et al. 1990 J
Immunol 145: 3100, G Cáceres-Dittmar et al. 1993 Clin
Exp Immunol 91: 500, S Frankenburg et al. 1993 Parasite
Immunol 15: 509) and a dominant T helper (Th)1-type (C Pirmez
et al. 1993 J Clin Invest 91: 1390) and/or CD8+ T cell
(AM Da-Cruz et al. 1994 Infect Immun 62: 2614)
responses associated with healing of the disease. In
contrast, the progression of DCL (HW Murray et al. 1984
J Immunol 133: 2250) or VL (EM Carvalho et al. 1985 J
Clin Invest 76: 2066, BJ Holaday et al. 1993 J Infect
Dis 167: 411) is related to markedly reduced lymphocyte
proliferation and decreased IL-2 and IFN-g production by
peripheral lymphocytes in response to leishmanial antigens.
Moreover, the IFN-g levels in children with asymptomatic and
subclinical self-healing L. chagasi infections were
significantly higher than those observed in children with
subclinical infections progressing to VL (EM Carvalho et
al. 1992 J Infect Dis 165: 536, Holaday et al. loc.
cit.).
VACCINE PERSPECTIVES
The solid immunity observed following convalescence to CL or
VL has suggested that vaccination may prove to be the most
cost-effective intervention method for the prevention and
control of the various leishmanial infections at a population
level. Vaccines consisting of viable forms of the pathogen
itself have been evaluated with CL in the Old World. Both
cellular and humoral immune response to leishmanial antigens
(MS Green et al. 1983 Parasite Immunol 5: 337) and
resistance to reinfection usually develop in subjects
vaccinated against CL using living L. major promastigotes
(reviewed in CL Greenblatt 1985 Leishmaniasis. K-P Chang, RS
Bray (eds). Elsevier 163 pp.). However, large scale human
trials have clearly indicated that protection requires prior
controlled induction of disease with a virulent parasite
(reviewed in AE Gunders 1987 The leishmaniasis in biology and
medicine. W Peters, R Killick-Kendrick (eds). Academic Press
928 pp.). As a consequence, this type of immunization can be
used only with Leishmania species that produce benign
self-healing lesions.
Prophylatic immunization using killed promastigote vaccine is
currently only in experimental stages. Unlike the mouse
studies (JG Howard et al. 1982 J Immunol 129: 2206),
periodic boosting in humans using inactivated parasites
promotes a delayed-type hypersensitivity (DTH) response to
leishmanial antigen, which seems to increase the recipient's
chance of being protected (W Mayrink et al. 1979 Trans
R Soc Trop Med Hyg 73: 385, E Nascimento et al. 1990
Infect Immun 58: 2198, M Castes et al. 1994 Vaccine 12:
1041). However, whether killed vaccine can achieve similar
levels of immunity as living vaccine is at present unknown.
Preliminary vaccination trials with killed organisms have
given conflicting results. Although the efficacy of a phenol
killed-promastigote vaccine showed an 82% protection rate
against American CL (SB Pessoa, BR Pestana 1941 Arch Hig Saude
Publ 6: 141), a similar approach failed to vaccinate against
Old World CL (DA Barberian 1944 Arch Dermatol Syphilol 50:
234). The first field trial evaluating the efficacy of a
polyvalent vaccine showed that more than 70% of the vaccinees
became skin positive, but the study was inconclusive by low
incidence rate in the control group (Mayrink et al.
loc. cit.). In a similar trial, only 30% of vaccines showed
skin test conversion; of those people that did convert, there
was about a 70% decrease in the incidence of natural disease
compared with the incidence in the control group (W Mayrink
et al. 1985 Ann Trop Med Parasitol 79: 259). In a third
trial in the Amazon region of Brazil, there were also
reductions (67.3 and 85.7%) in the annual incidence of CL
among vaccinees developing a positive leishmanin skin test.
However, when the skin test-positive and skin test-negative
vaccines in that study were combined, the difference between
the vaccinated and control groups was not significant (CMF
Antunes et al. 1986 Int J Epidemiol 15: 572). Other
studies have shown that development of DTH response may not be
necessarily accompanied by protection, following vaccination
with attenuated promastigotes (PEC Manson-Bahr 1961 Trans R
Soc Trop Med Hyg 55: 550, J Salazar 1965 Arch Venez Med Trop
Parasitol Med 5: 365, D Heyneman 1971 Bull WHO 44: 499). DTH
may also be a marker that is frequently linked with, while not
itself being, the underlying mechanism of pathogenesis in L.
braziliensis infection (NG Saravia et al. 1989 J Infect
Dis 159: 725).
Over the last ten years much effort has been devoted to the
development of standardized and safe vaccines that should be
able to produce long-lasting immunity against all types of
human leishmaniasis. Prospects for human immunoprophylaxis
with a new generation of safe and effective subunit vaccines
(using either recombinant or synthetic peptides or infectious
recombinant vectors) is now within our reach (DM Yang et
al. 1990 J Immunol 145: 2281, A Jardim et al. 1991
J Exp Med 172: 645, D McMahon-Pratt et al. 1993 Infect
Immun 61: 3351). In order to review the current status of
vaccine development towards the control of human
leishmaniasis, the WHO Special Program for Research and
Training on Tropical Diseases (TDR), the Pan American Health
Organization (PAHO), the National Health Foundation, Brazilian
Ministry of Health (FNS/MS), and the Federal University of
Bahia (UFBA), jointly organized two meetings in Salvador,
Bahia (Brazil).
FIRST WORKSHOP
The first Workshop (on "Vaccine Efficacy Trials Against
Leishmaniasis and Preparation of Phase III Protocols") aimed
to review the progress made in various studies using crude
killed promastigote antigens and plan future activities on
field efficacy trials (Phase III) for candidate vaccines. The
group reviewed general issues such as epidemiological aspects
of the disease in several Latin American countries, as well as
safety and regulatory procedures employed on vaccine studies
[the WHO has prepared specific guidelines for evaluating
vaccine, which include recommendations for field trials that
address questions of design, selection of study populations,
execution of the trials, data analysis, and ethical principles
with regard to safety, protection, and benefits for
individuals (WHO 1992 Tech Rep Ser, 822)]. The discussion was
focused on (i) vaccine development program in the Old World
with killed L. major produced by the Razi Institute, Iran
(ii); previous South American human vaccination trials using
an intramuscular injection of merthiolate-treated organisms
made of a mixture of Leishmania strains; (iii) canine
vaccination trials (using crude promastigote antigens) in
Brazil and France; (iv) Phase I and II trials with the
Brazilian Good Manufacturing Practice (GMP) produced one
strain vaccine (merthiolate-treated promastigotes of L.
amazonensis); and (v) other human leishmanial vaccine (killed
promastigotes plus BCG) trials developed in Venezuela and
Ecuador. Studies in Brazil (Dr SCF Mendonca and co-workers,
this meeting) and Venezuela (Castes et al. loc. cit., M
Castes, this meeting) have shown that a high proportion of
healthy volunteers immunized with inactivated Leishmania
promastigotes (with or without BCG) converted for T-cell
responder phenotypes to parasite antigen, but vaccination
consistently elicited low IFN-g production from T lymphocytes
of vaccinees. Whether the intensity or duration of the
elicited responses and their roles in vaccine-induced immunity
may vary according to the antigenic composition of the
injected strain (or the quality control of the vaccine
materials), or the genetic variability of the host remains to
be established. In addition, field trials in Venezuela have
shown that anti-leishmanial antibody responses were not
enhanced in the two vaccine groups receiving killed
promastigotes (with/without BCG) compared with the BCG alone
and placebo groups. However, all vaccine groups showed a
pattern of immune response consistent with either a response
to the skin-test antigen or, more likely, seasonal endemic
exposure to leishmanial infection (CE Sharples et al.
1994 Vaccine 12: 1403). Future field studies (Phase III trials
will be funded by WHO/TDR) are planned for testing killed
Leishmania vaccine in several endemic foci in the Americas.
Such studies are important since there is still much to be
done for assessing the effectiveness of vaccination in the
absence of natural challenge. The artifical induction of
acquired resistance will also depend on detailed knowledge of
the natural history of the human host-parasite relationship
occurring in each endemic area. The group suggested that the
WHO/PAHO should take an active part in the New World
leishmaniasis vaccine program.
SECOND WORKSHOP
Participants at the second Workshop (on "Development of the
Second Generation Vaccine Against Leishmaniasis") reviewed the
latest strategies in vaccine design against leishmanial
infections. Current research employing mouse models is
providing the foundation for studies designed not only to
identify leishmanial protective immunogens, but also to
provide a better understanding of the immune mechanisms (by
correlating specific immunologic responses with protection)
responsible for solid immunity in vaccinated animals.
Attention was focused on the induction of immunoprotection
using (i) purified leishmanial antigenic components such as
the major cell surface glycoprotein gp63 of Leishmania (DG
Russell, J Alexander 1988 J Immunol 140: 1274, LP Kahl et
al. 1989 J Immunol 142: 4441); the promastigote surface
glycoprotein gp46 of L. amazonensis (J Champsi, D
McMahon-Pratt 1988 Infect Immun 56: 3272) or related
polypeptyde antigens PSA-2 (JM Burns et al. 1991 J
Immunol 146: 742, Dr E Handman and coworkers, this meeting);
the pure protein dp72 of L. donovani (N Rachamim, CL Jaffe
1993 J Immunol 150: 2322); the glycolipid LPG (E Handman, GF
Mitchell 1985 Proc Natl Acad Sci 82: 5910, MJ McConville et
al. 1987 Proc Natl Acad Sci USA 84: 8941) or the
lipophosphoglycan-associated membrane proteins LPG-AP/KMP-11
of Leishmania (DM Russo et al. 1992 J Immunol 148: 202,
A Jardim et al. 1995 Biochem J 305: 307); and the
amastigote proteins P-2/A-2, P-4, and P-8 of L. pifanoi (Dr D
McMahon-Pratt and coworkers, this meeting); or (ii) Leishmania
antigens synthetized through gene cloning such as the rp63
(LL Button, WR McMaster 1988 EMBO J 7: 93, DG Russel, J
Alexander loc. cit.), rp24-LACK (Dr N Glaichenhaus, this
meeting) and rLeIF (Dr YAW Skeiky and co-workers, this
meeting) leishmanial recombinant proteins; or (iii) Leishmania
mimicked antigens by synthetic peptides such as the gp63/pt-3
and gp63/pt-6 T cell epitopes (A Jardim et al. 1990 J
Exp Med 172: 645). Complete or appreciable levels of
protection against CL and/or VL have been achieved in
genetically resistant and susceptible strains of mice by using
these candidate vaccines, either in conjunction with adjuvants
(Handman, Mitchell loc. cit., McConville et al. loc.
cit., Champsi, McMahon-Pratt loc. cit., Russel, Alexander
loc. cit., Rachamim, Jaffe loc. cit.) or delivered in vivo by
infectious multivaccine expression vectors such as the
attenuated recombinant Salmonella/gp63 (Yang et al.
loc. cit.), BCG/gp63 (Dr WR McMaster and co-workers, this
meeting), or Vaccinia/gp63 (McMahon-Pratt et al. loc.
cit.). Recent emphasis has also turned to the evaluation of
defined leishmanial antigens in humans to identify epitopes
that induce appropriate T-cell responses (MG Zallis et
al. 1988 Mem Inst Oswaldo Cruz 83: 117, SG Reed et
al. 1990 J Clin Invest 85: 690, JM Burns et al.
1991 J Immunol 146: 742, M Kemp et al. 1991 Scand J
Immunol 33: 219, SCF Mendonca et al. 1991 Clin Exp Med
83: 472, DM Russo et al. 1991 J Immunol 147: 3575, DM
Russo et al. 1992 J Immunol 148: 202). Either the
recombinant or native form of gp63, for instance, is a strong
T-cell immunogen (capable of inducing CD4+ T cell
proliferative response and IFN-g production) in leishmaniasis
patients (Mendonca et al. loc. cit., Russo et
al. loc. cit.). Moreover, induction of protection using a
potent vasodilator termed erytheme-inducing factor (which can
neutralize the immunosupressive effect of salive) present in
sandfly salive (RG Titus, JMC Ribeiro 1988 Science 239: 1306,
JMC Ribeiro et al. 1990 Br J Pharmacol 101: 932, JMC
Ribeiro et al. 1993 Science 260: 539), or the salivary
immunosupressive protein itself (Dr J David, this meeting) as
vaccine is now within our reach. Alternatively, the recent
successful immunization of susceptible strains of mice (RG
Titus et al. 1995 Proc Natl Acad Sci USA, in press)
using genetically avirulent [parasites lacking the
dihydrofolate reductase-thymidylate synthetase, or dhfr-ts by
gene replacement (A Cruz et al. 1991 Proc Natl Acad Sci
USA 88: 7170)] L. major live vaccine has provided a
revolutionary new approach in vaccinology. The ability of
dhfr-ts- parasites to differentiate and persist briefly (for
up to two months, declining with a half-life of about 2-3
days) may reflect a locus-specific advantage of dhfr-ts-
knockouts in prolonging the period and diversity of antigen
delivery by Leishmania (Titus et al. loc. cit.).
Potentially, dhfr-ts- could be used as a delivery system for
other antigen and/or adjuvant (e.g., IL-12).
CONCLUDING REMARKS
At the conclusion of the meeting the participants discussed
about general problems encountered and future development.
Attention was focused on (i) the value of killed promastigotes
as vaccinating materials, and whether a single strain or
defined antigen vaccine could induce heterologous protection;
and (ii) how to measure vaccine effectiveness (the degree and
duration of the protection) in humans without an actual
challenge infection. Although many advances in understanding
some of the cellular immune responses generated during
leishmanial infections have been made, there is still many
unanswered questions about the complex immunologic mechanisms
involved in the control of human leishmaniasis. Furthermore,
vaccine-induced immunity against leishmanial infection in
murine models have the fundamental drawback of an uncertain
correlation with human infection. However, as non-human
primate host responses to Leishmania (VA Dennis et al.
1986 Exp Parasitol 61: 319, JI Githure et al. 1986
Trans R Soc Trop Med Hyg 80: 575, R Lujan et al. 1986
Am J Trop Med Hyg 35: 1103, I Vouldoukis et al. 1986 J
Parasitol 72: 472, OJ Pung, RE Kuhn 1987 J Med Primatol 16:
165, JO Olobo et al. 1992 Scand J Immunol 36: 48) are
very similar to those observed in humans, primate models could
provide an indication of the potential success and/or
limitations for human vaccine against leishmaniasis. This will
be especially important in the case of L. braziliensis complex
parasites where the monkey models studied to date are
susceptible to infection (R Lujan et al. 1986 Exp
Parasitol 61: 348, R Lujan et al. 1990 J Parasitol 76:
594, FT Silveira et al. 1989 Rev Soc Bras Med Trop 22:
125, FT Silveira et al. 1990 Rev Inst Med Trop S Paulo
32: 387) and the murine model is limited. The parallels found
between humans and other primates in susceptibility,
clinicopathologic changes, and immunologic responses to
leishmanial infections is not suprising, given their close
phylogenetic and immunologic relationship (NL Letvin et
al. 1983 Eur J Immunol 13: 345, H Kishino, M Hasegawa
1990 Methods Enzymol 183: 550, J Klein et al. 1993
Immunol Rev 11: 213). Success of a vaccine depends not only on
the identification of protective antigens but also the
adoption of a suitable vaccination protocol. For vaccination,
the issues are route of immunization, doses, parasite stage,
timing between vaccination and challenge. Of special interest
will be studies using primates for delineating antigens or
delivery systems (adjuvants/infectious agents, or a new
construct/cocktail vaccine) relevant for protective immunity
in humans.
Copyright 1995 Fundacao Oswaldo Cruz
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