Rationale and plans for developing a non-replicating, metabolically active, radiation-attenuated Plasmodium falciparum sporozoite vaccine
1 Uniformed Services University of the Health Sciences, Bethesda, MD 20814,
USA
2 Sanaria Inc., Rockville, MD 20852, USA
* Author for correspondence (e-mail: slhoffman{at}sanaria.com)
Accepted 17 July 2003
![]() |
Summary |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Three practical questions must be addressed before manufacturing for clinical trials: (1) can one administer the vaccine by a route that is clinically practical; (2) can one produce adequate quantities of sporozoites; and (3) can sporozoites be produced with the physical characteristics that meet the regulatory, potency and safety requirements of regulatory authorities? Once these questions have been answered, Sanaria will demonstrate that the vaccine protects >90% of human recipients against experimental challenge with Pf sporozoites, can be produced with an efficiency that makes it economically feasible, and protects >90% of African infants and children from infection, and thus from severe morbidity and mortality. By producing a vaccine for travelers, Sanaria will provide the infrastructure, regulatory foundation and funds necessary to speed licensure, manufacturing and deployment of the vaccine for the infants and children who need it most.
Key words: malaria vaccine, immunization, Plasmodium falciparum, radiation-attenuated sporozoite, irradiated sporozoite
![]() |
Introduction |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The promise of impending success was short lived and the reasons for failure were multi-factorial. The parasites grew increasingly resistant to highly effective and affordable anti-malarial medications, vector control measures lapsed, and transmigration, war and economic disruption became increasingly more common in endemic areas of the developing world. As a result, falciparum malaria has resurged, annually placing 2.5 billion humans at risk, causing 300-900 million infections and killing 1-3 million people. Of the many social, economic, environmental and political problems that afflict the developing world, falciparum malaria is increasingly seen as both a root cause and cruel result of these inequities and is a singular impediment to solving these complex problems. Controlling falciparum malaria in the developing world may be possible without an effective vaccine. In practice, given social, political and economic realities, we believe that a vaccine may be an essential component of a sustainable control program and will be required for a global eradication campaign.
It is in this context that the modern period of malaria vaccine development
has been particularly frustrating. Since the early 1980s, breathtaking
technological advances in molecular biology and medical science have occurred.
These advances accelerated the identification of stage-specific P.
falciparum proteins and epitopes and host immune mechanisms and
responses. This knowledge was translated into a range of novel vaccine
candidates (Richie and Saul,
2000; Long and Hoffman,
2002
). In one sense, this modern period has been the golden age of
malaria vaccine research and human testing. However, in spite of the herculean
efforts of malaria researchers, the majority of these vaccines has failed to
provide any protective immunity in humans - with only one demonstrating
reproducible short-term protection against infection in 40-70% of recipients
(Stoute et al., 1998
;
Kester et al., 2001
;
Bojang et al., 2001
).
Given enough time and resources, these vaccine strategies, or others yet to
be developed, may ultimately lead to a robust vaccine. However, at a recent
Keystone meeting, `Malaria's Challenge: From Infants to Genomics to Vaccines'
(Long and Hoffman, 2002), the
attendees were polled as to when they thought a malaria vaccine might be
`launched' as a commercial product. Many in the room indicated that they
thought the first vaccine would not be launched until 2016-2025. The leader of
GlaxoSmithKline (GSK)'s efforts to develop a recombinant P.
falciparum circumsporozoite protein (PfCSP) vaccine voiced the most
optimism. It was indicated that, if all went well, this single protein vaccine
could be `launched' in 7-8 years(2009-2010). Given that GSK and the US Army
have been working on a recombinant protein vaccine since the 1984 cloning of
the PfCSP (Dame et al., 1984
)
and that many malariologists express concern as to whether a single protein
vaccine will be adequate to sustainably control malaria, this time line of
>25 years for development of a single protein vaccine places a chillingly
realistic perspective on the possibilities for developing vaccines that will
truly reduce the burden of this disease.
![]() |
Protective immunity after immunization with radiation-attenuated sporozoites |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Potential solutions to these problems, although not necessarily recognized
at the time as being related to developing an attenuated sporozoite vaccine,
were being reported. In 1975, a method for culturing P. falciparum in
vitro was reported (Trager and
Jensen, 1976; Haynes et al.,
1976
), followed in 1982 by a method for producing gametocytes from
these cultures (Campbell et al.,
1982
). In 1986, it was reported that humans could be infected by
the sporozoites produced in mosquitoes that had fed on these in vitro
cultures (Chulay et al., 1986
).
There was therefore a way to produce sporozoites without the difficulties of
in vivo production of gametocytes in humans.
However, by that time, several promising developments launched the modern
era of malaria subunit vaccine development. A monoclonal antibody against the
major surface protein of sporozoites, the circumsporozoite protein (CSP), had
been produced and shown to protect mice in passive transfer experiments
(Yoshida et al., 1980).
Additionally, the gene encoding the PfCSP protein had been cloned and
sequenced (Dame et al., 1984
).
Coincidentally, the first purified recombinant protein vaccine, the hepatitis
B surface antigen vaccine, was developed and marketed
(Hilleman, 1987
). The weight
of evidence and trends in vaccine science seemed to offer malaria researchers
a roadmap to quickly develop a human malaria vaccine. Returning to an
attenuated whole-parasite vaccine seemed unnecessary and dated, and all
subsequent efforts focused on the promise of subunit vaccines.
In 1987, when the first recombinant protein
(Ballou et al., 1987) and
synthetic peptide (Herrington et al.,
1987
) vaccines did not prove to be as protective as expected,
instead of considering the development of an attenuated sporozoite vaccine,
scientists focused on understanding the immune mechanisms responsible for
protective immunity, and the antigenic targets of these protective immune
responses, and developing subunit vaccines and vaccine delivery systems that
induced such protection. Much of this basic work was carried out in the P.
berghei and P. yoelii rodent model systems. This rodent malaria
work provided important insights into irradiated sporozoite vaccine-induced
protection and led to the development of a number of candidate vaccines
(Nussenzweig and Nussenzweig,
1989
; Hoffman et al.,
1996
; Hoffman and Miller,
1996
). Importantly, in stark contrast to subunit vaccine
formulations, the protective results of rodent and human irradiated sporozoite
studies have been strikingly concordant.
In 1989, after a number of disappointing clinical trials of subunit PfCSP
vaccines, immunization of volunteers with gamma radiation-attenuated
Pf sporozoites was begun at the Naval Medical Research Center and
Walter Reed Army Institute of Research. The goal of this research was to
better delineate the clinical characteristics and requirements that led to
protecting humans with the irradiated sporozoite vaccine, to assess the
protective immune responses elicited in humans and to identify the antigens
and epitopes on those proteins that elicited immune responses in humans.
Preliminary clinical results and extensive immunological assay results from
these studies were published (Egan et al.,
1993; Malik et al.,
1991
; Wizel et al.,
1995a
,b
;
Krzych et al., 1995
; Doolan et
al., 1997
,
2000
). These immunological
studies, combined with those of others on this subject
(Herrington et al., 1991
;
Edelman et al., 1993
; Nardin
et al., 1989
,
1990
;
Nardin, 1990
; Moreno et al.,
1991
,
1993
), increased our
understanding of the immunological responses in humans immunized with
radiation-attenuated P. falciparum sporozoites.
![]() |
Foundation for current plans |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1. There was a dose response in regard to protective immunity among
volunteers challenged by the bite of 5-14 infected mosquitoes
Thirteen of 14 volunteers (93%) immunized by the bites of >1000
infected, irradiated mosquitoes were protected against developing blood-stage
P. falciparum infection when challenged within 10 weeks of their last
primary immunization; there were 35 challenges of these volunteers and there
was complete protection against development of blood-stage infection in 33 of
the 35 challenges (94%).
Four of 10 volunteers (40%) immunized by the bite of >200 and <1000 infected, irradiated mosquitoes were protected against developing blood-stage P. falciparum infection when challenged within 10 weeks of their last primary immunization, a significantly lower level of protective immunity than among volunteers immunized with >1000 infective bites (P=0.0088; Fisher's exact test, two-tailed); there were 15 challenges of these volunteers and there was complete protection against development of blood-stage infection in five of the 15 challenges (33%), a significantly lower level of protective immunity than among volunteers immunized with >1000 infective bites (P=0.000015; Fisher's exact test, two-tailed).
2. Protective immunity lasted for at least 42 weeks (10.5
months)
Five of six of the above 14 volunteers when challenged 23-42 weeks (23, 36,
39, 41 and 42 weeks) after their last primary or secondary immunization were
protected against experimental challenge. Except for a single challenge of one
volunteer five years after their last immunization (not protected), there were
no other challenges assessing longevity of protective immunity.
3. Protection was not strain specific
Four volunteers were challenged with isolates of P. falciparum
that were different from the isolates with which they were immunized. The four
volunteers were completely protected in seven of seven such challenges with
different isolates of P. falciparum.
Thus, protection was achieved in >90% of challenge experiments after >1000 mosquito bites, lasted for at least 10.5 months and was not P. falciparum isolate (strain) specific.
![]() |
Back to the future |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We concluded that there were three critical questions that had to be answered before moving into cGMP manufacturing and clinical trials:
Can one administer the vaccine by a route that is clinically
practical?
In the past, humans have been immunized by exposure to irradiated
Anopheles mosquitoes infected with P. falciparum or P.
vivax sporozoites. Generally, mice have been immunized by intravenous
administration of P. berghei or P. yoelii sporozoites. It is
not practical to consider large-scale immunization by the bite of infected
mosquitoes, and there are no vaccines used as public health measures that are
administered intravenously. Essentially, all vaccines are administered by
intradermal, subcutaneous or intramuscular routes. Work is in progress to
demonstrate that this is feasible for irradiated sporozoites.
Can one produce adequate quantities of sporozoites?
It requires the bites of 1000 irradiated infected mosquitoes to
consistently protect humans (Hoffman et
al., 2002). Most entomologists believe that each mosquito injects
only 10-20 sporozoites but may inject up to 100 sporozoites
(Beier et al., 1991
).
Therefore, the bites of 1000 infected mosquitoes inoculating 10-100
sporozoites per mosquito bite = 104-105 sporozoites
delivered for a full immunization regimen. Most laboratories produce
2x104 P. falciparum sporozoites per mosquito. We and
others are now working on producing many more sporozoites per mosquito. If one
could produce >105 P. falciparum sporozoites per
mosquito, a single infected mosquito could provide all the sporozoites needed
for a full human immunization regimen.
Can sporozoites be produced with the physical characteristics that
meet regulatory, potency and safety requirements to be a licensed
vaccine?
A process for manufacturing a vaccine in mosquitoes that meets regulatory
requirements will require development and optimization of: (1) a process that
yields aseptic, purified, potent sporozoites; (2) a process for optimal
radiation attenuation of cryopreserved sporozoites and (3) validated lot
release assays that indicate that aseptic, purified, optimally attenuated and
cryopreserved sporozoites are potent and adequately attenuated (safe). Sanaria
is working on developing and optimizing all three.
![]() |
Factors that will speed licensing a malaria vaccine that prevents infection |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Rationale for why an irradiated sporozoite vaccine will reduce malaria-associated severe disease and mortality in infants and children in Africa |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
Conclusion |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The opinions and assertions herein are the private ones of the authors and are not to be construed as official or reflecting the views of the US Navy or the Department of Defense.
![]() |
References |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Baird, J. K., Owusu, A. S., Utz, G. C., Koram, K., Barcus, M.
J., Jones, T. R., Fryauff, D. J., Binka, F. N., Hoffman, S. L. and Nkrumah, F.
N. (2002). Seasonal malaria attack rates in infants and young
children in northern Ghana. Am. J. Trop. Med. Hyg.
66,280
-286.
Ballou, W. R., Hoffman, S. L., Sherwood, J. A., Hollingdale, M. R., Neva, F. A., Hockmeyer, W. T. and Gordon, D. M. (1987). Safety and efficacy of a recombinant DNA Plasmodium falciparum sporozoite vaccine. Lancet 1,1277 -1281.[Medline]
Beier, J. C., Davis, J. R., Vaughan, J. A., Noden, B. H. and Beier, M. S. (1991). Quantitation of Plasmodium falciparum sporozoites transmitted in vitro by experimentally infected Anopheles gambiae and Anopheles stephensi. Am. J. Trop. Med. Hyg. 44,564 -570.[Medline]
Beier, J. C., Oster, C. N., Onyango, F. K., Bales, J. D., Sherwood, J. A., Perkins, P. V., Chumo, D. K., Koech, D. V., Whitmire, R. E., Roberts, C. R. et al. (1994). Plasmodium falciparum incidence relative to entomologic inoculation rates at a site proposed for testing malaria vaccines in western Kenya. Am. J. Trop. Med. Hyg. 50,529 -536.[Medline]
Bojang, K. A., Milligan, P. J., Pinder, M., Vigneron, L., Allouche, A., Kester, K. E., Ballou, W. R., Conway, D. J. and Reece, W. H. (2001). Efficacy of RTS,S/AS02 malaria vaccine against Plasmodium falciparum infection in semi-immune adult men in The Gambia: a randomised trial. Lancet 358,1927 -1934.[CrossRef][Medline]
Campbell, C. C., Collins, W. E., Nguyen-Dinh, P., Barber, A. and Broderson, J. R. (1982). Plasmodium falciparum gametocytes from culture in vitro develop to sporozoites that are infectious to primates. Science 217,1048 -1050.[Medline]
Chulay, J. D., Schneider, I., Cosgriff, T. M., Hoffman, S. L., Ballou, W. R., Ouakyi, I. A., Carter, R., Trosper, J. H. and Hockmeyer, W. T. (1986). Malaria transmitted to humans by mosquitoes infected from cultured Plasmodium falciparum. Am. J. Trop. Med. Hyg. 35,66 -68.[Medline]
Church, L. W., Le, T. P., Bryan, J. P., Gordon, D. M., Edelman, R., Fries, L., Davis, J. R., Herrington, D. A., Clyde, D. F., Shmuklarsky, M. J. et al. (1997). Clinical manifestations of Plasmodium falciparum malaria experimentally induced by mosquito challenge. J. Infect. Dis. 175,915 -920.[Medline]
Clyde, D. F. (1990). Immunity to falciparum and vivax malaria induced by irradiated sporozoites: a review of the University of Maryland studies, 1971-75. Bull WHO 68, 9-12.[Medline]
Clyde, D. F., McCarthy, V. C., Miller, R. M. and Hornick, R. B. (1973b). Specificity of protection of man immunized against sporozoite-induced falciparum malaria. Am. J. Med. Sci. 266,398 -401.[Medline]
Clyde, D. F., McCarthy, V. C., Miller, R. M. and Woodward, W. E. (1975). Immunization of man against falciparum and vivax malaria by use of attenuated sporozoites. Am. J. Trop. Med. Hyg. 24,397 -401.[Medline]
Clyde, D. F., Most, H., McCarthy, V. C. and Vanderberg, J. P. (1973a). Immunization of man against sporozoite-induced falciparum malaria. Am. J. Med. Sci. 266,169 -177.[Medline]
Dame, J. B., Williams, J. L., McCutchan, T. F., Weber, J. L., Wirtz, R. A., Hockmeyer, W. T., Maloy, W. L., Haynes, J. D., Schneider, I., Roberts, D. et al. (1984). Structure of the gene encoding the immunodominant surface antigen on the sporozoite of the human malaria parasite Plasmodium falciparum. Science 225,593 -599.[Medline]
Doolan, D. L., Hoffman, S. L., Southwood, S., Wentworth, P. A., Sidney, J., Chestnut, R. W., Keogh, E., Apella, E., Nutman, T. B., Lal, A. A. et al. (1997). Degenerate cytotoxic T cell epitopes from P. falciparum restricted by HLA-A and HLA-B supertypes alleles. Immunity 7,97 -112.[Medline]
Doolan, D. L., Southwood, S., Chesnut, R., Appella, E., Gomez,
E., Richards, A., Higashimoto, Y. I., Maewal, A., Sidney, J., Gramzinski, R.
A. et al. (2000). HLA-DR-promiscuous T cell epitopes from
Plasmodium falciparum pre-erythrocytic-stage antigens restricted by multiple
HLA class II alleles. J. Immunol.
165,1123
-1137.
Edelman, R., Hoffman, S. L., Davis, J. R., Beier, M., Sztein, M. B., Losonsky, G., Herrington, D. A., Eddy, H. A., Hollingdale, M. R., Gordon, D. M. et al. (1993). Long-term persistence of sterile immunity in a volunteer immunized with X-Irradiated Plasmodium falciparum sporozoites. J. Infect. Dis. 168,1066 -1070.[Medline]
Egan, J. E., Hoffman, S. L., Haynes, J. D., Sadoff, J. C., Schneider, I., Grau, G. E., Hollingdale, M. R., Ballou, W. R. and Gordon, D. M. (1993). Humoral immune responses in volunteers immunized with Irradiated Plasmodium falciparum sporozoites. Am. J. Trop. Med. Hyg. 49,166 -173.[Medline]
Hale, B. R and Hoffman, S. L. (2003). A randomized, double-blind, placebo-controlled dose ranging trial of tafenaquine for weekly prophylaxis against Plasmodium falciparum. Clin. Infect. Dis. 36,541 -549.[CrossRef][Medline]
Haynes, J. D., Diggs, C. L., Hines, F. A. and Desjardins, R. E. (1976). Culture of human malaria parasites Plasmodium falciparum. Nature 263,767 -769.[Medline]
Herrington, D. A., Clyde, D. F., Losonsky, G., Cortesia, M., Murphy, J. R., Davis, J., Bager, S. and Felix, A. M. (1987). Safety and immunogenicity in man of a synthetic peptide malaria vaccine against Plasmodium falciparum sporozoites. Nature 328,257 -259.[CrossRef][Medline]
Herrington, D., Davis, J., Nardin, E., Beier, M., Cortese, J., Eddy, H., Losonsky, G., Hollingdale, M., Sztein, M., Levine, M. et al. (1991). Successful immunization of humans with Irradiated sporozoites: humoral and cellular responses of the protected individuals. Am. J. Trop. Med. Hyg. 45,539 -547.[Medline]
Hilleman, M. R. (1987). Yeast recombinant hepatitis B vaccine. Infection 15, 3-7.[Medline]
Hoffman, S. L. (1997). Experimental challenge
of volunteers with malaria. Ann. Intern. Med.
127,233
-235.
Hoffman, S. L. and Miller, L. H. (1996). Perspectives on malaria vaccine development. In Malaria Vaccine Development: A Multi-Immune Response Approach (ed. S. L. Hoffman), pp. 1-13. Washington, DC: ASM Press.
Hoffman, S. L., Franke, E. D., Hollingdale, M. R. and Druilhe, P. (1996). Attacking the infected hepatocyte. In Malaria Vaccine Development: A Multi-Immune Response Approach (ed. S. L. Hoffman), pp. 35-75. Washington, DC: ASM Press.
Hoffman, S. L., Goh, L. M., Luke, T. C., Schneider, I., Le, T. P., Doolan, D. L., Sacci, J., de la Vega, P., Dowler, M., Paul, C. et al. (2002). Protection of humans against malaria by immunization with radiation-attenuated Plasmodium falciparum sporozoites. J. Infect. Dis. 185,1155 -1164.[CrossRef][Medline]
Hoffman, S. L., Oster, C. N., Plowe, C. V., Woollett, G. R., Beier, J. C., Chulay, J. D., Wirtz, R. A., Hollingdale, M. R. and Mugambi, M. (1987). Naturally acquired antibodies to sporozoites do not prevent malaria: vaccine development implications. Science 237,639 -642.[Medline]
Kester, K. E., McKinney, D. A., Tornieporth, N., Ockenhouse, C. F., Heppner, D. G., Hall, T., Krzych, U., Delchambre, M., Voss, G., Dowler, M. G. et al. (2001). Efficacy of recombinant circumsporozoite protein vaccine regimens against experimental Plasmodium falciparum malaria. J. Infect. Dis. 183,640 -647.[CrossRef][Medline]
Krzych, U., Lyon, J. A., Jareed, T., Schneider, I., Hollingdale, M. R., Gordon, D. M. and Ballou, W. R. (1995). T lymphocytes from volunteers immunized with Irradiated Plasmodium falciparum sporozoites recognize liver and blood stage malaria antigens. J. Immunol. 155,4072 -4077.[Abstract]
Long, C. A. and Hoffman, S. L. (2002).
Parasitology: malaria - from infants to genomics to vaccines.
Science 297,345
-347.
Malik, A., Egan, J. E., Houghten, R. A., Sadoff, J. C. and Hoffman, S. L. (1991). Human cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein. Proc. Natl. Acad. Sci. USA 88,3300 -3304.[Abstract]
McCarthy, V. C. and Clyde, D. F. (1977). Plasmodium vivax: correlation of circumsporozoite precipitation (CSP) reaction with sporozoite-induced protective immunity in man. Exp. Parasitol. 41,167 -171.[Medline]
Moreno, A., Clavijo, P., Edelman, R., Davis, J., Sztein, M., Herrington, D. and Nardin, E. (1991). Cytotoxic CD4+ T cells from a sporozoite-immunized volunteer recognize the Plasmodium falciparum CS protein. Int. Immunol. 3,997 -1003.[Abstract]
Moreno, A., Clavijo, P., Edelman, R., Davis, J., Sztein, M.,
Sinigaglia, F. and Nardin, E. (1993). CD4+ T cell clones
obtained from Plasmodium falciparum sporozoite-immunized volunteers recognize
polymorphic sequences of the circumsporozoite protein. J.
Immunol. 151,489
-499.
Nardin, E. H. (1990). T cell responses in a sporozoite-immunized human volunteer and a chimpanzee. Immunol. Lett. 25,43 -48.[CrossRef][Medline]
Nardin, E. H., Herrington, D. A., Davis, J., Levine, M., Stuber, D., Takacs, B., Caspers, P., Barr, P., Altszuler, R., Clavijo, P. et al. (1989). Conserved repetitive epitope recognized by CD4+ clones from a malaria-immunized volunteer. Science 246,1603 -1606.[Medline]
Nardin, E. H., Nussenzweig, R. S., Altszuler, R., Herrington, D., Levine, M., Murphy, J., Davis, J., Bathurst, I., Barr, P., Romero, P. et al. (1990). Cellular and humoral immune responses to a recombinant P. falciparum CS protein in sporozoite-immunized rodents and human volunteers. Bull. WHO 68, 85-87.[Medline]
Nussenzweig, R. S., Vanderberg, J., Most, H. and Orton, C. (1967). Protective immunity produced by the injection of X-irradiated sporozoites of Plasmodium berghei. Nature 216,160 -162.[Medline]
Nussenzweig, V. and Nussenzweig, R. S. (1989). Rationale for the development of an engineered sporozoite malaria vaccine. Adv. Immunol. 45,283 -334.[Medline]
Owusu-Agyei, S., Koram, K. A., Baird, J. K., Utz, G. C., Binka,
F. N., Nkrumah, F. K., Fryauff, D. J. and Hoffman, S. L.
(2001). Incidence of symptomatic and asymptomatic Plasmodium
falciparum infection following curative therapy in adult residents of northern
Ghana. Am. J. Trop. Med. Hyg.
65,197
-203.
Richie, T. L. and Saul, A. (2000). Progress and challenges for malaria vaccines. Nature 415,694 -701.[CrossRef]
Rieckmann, K. H. (1990). Human immunization with attenuated sporozoites. Bull WHO 68, 13-16.[Medline]
Rieckmann, K. H., Beaudoin, R. L., Cassells, J. S. and Sell, D. W. (1979). Use of attenuated sporozoites in the immunization of human volunteers against falciparum malaria. Bull WHO 57,261 -265.[Medline]
Rieckmann, K. H., Carson, P. E., Beaudoin, R. L., Cassells, J. S. and Sell, K. W. (1974). Sporozoite induced immunity in man against an Ethiopian strain of Plasmodium falciparum. Trans. R. Soc. Trop. Med. Hyg. 68,258 -259.[Medline]
Stoute, J. A., Kester, K. E., Krzych, U., Wellde, B. T., Hall, T., White, K., Glenn, G., Ockenhouse, C. F., Garcon, N., Schwenk, R. et al. (1998). Long-term efficacy and immune responses following immunization with the RTS, S malaria vaccine. J. Infect. Dis. 178,1139 -1144.[Medline]
Trager, W. and Jensen, J. B. (1976). Human malaria parasites in continuous culture. Science 193,673 -675.[Medline]
Wizel, B., Houghten, R. A., Parker, K., Coligan, J. E., Church, P., Gordon,D. M., Ballou, W. R. and Hoffman, S. L. (1995a). Irradiated sporozoite vaccine induces HLA-B8-restricted cytotoxic T lymphocyte responses against two overlapping epitopes of the Plasmodium falciparum surface sporozoite protein 2. J. Exp. Med. 182,1435 -1445.[Abstract]
Wizel, B., Houghten, R., Church, P., Tine, J. A., Lanar, D. E., Gordon, D. M., Ballou, W. R., Sette, A. and Hoffman, S. L. (1995b). HLA-A2-restricted cytotoxic T lymphocyte responses to multiple Plasmodium falciparum sporozoite surface protein 2 epitopes in sporozoite-immunized volunteers. J. Immunol. 155,766 -775.[Abstract]
Yoshida, N., Nussenzweig, R. S., Potocnjak, P., Nussenzweig, V. and Aikawa, M. (1980). Hybridoma produces protective antibodies directed against the sporozoite stage of malaria parasite. Science 207,71 -73.[Medline]