726 40th FORUM IN IM~.z!UrgOLOG Y

timalarial immunity is largely T-independent" by "antimalarial immunity is subtly T-regulated".

References

Claassen, E. (1991), in "38th Forum in Immunology". Res. lmmunol., 142, 315-372.

Chougnet, C., Troye-Blomberg, M., Deloron, P., Kabi- lan, L., Lepers, J.P., Savel, J. & Perlmann, P. (1991), Human immune response in Plasmodiumfalciparum malaria. Synthetic peptides corresponding to known epitopes of the Pf 155/RESA antigen induce produc- tion of parasite-specific antibodies in vitro. J. Im- munol., 147 (in press).

DeFrance, T., Vanberuliet, B., Aubry, J., Takabe, Y., Arai, N., Miyajima, A., Yokota, T., Lee, F., Arai, K., DeVries, J.E. & Banchereau, B. (1987), B cell growth-promoting activity of recombinant IL4. J. Im- munol., 139, 1135-1139.

Haanen, J.B.A.G., de Waal Malefijt, R., Res, P.C.M., Kraakman, E.M., Ottenhoff, T.H.M., de Vries, R.R.P. & Spits, H. (1991), Selection of human T hel- per type-l-like T cell subset by mycobacteria. J. exp. Med., 174, 583-592.

Jenilek, D.F. & Lipsky, P.E. (1988), Inhibitory influence of IL4 on human B responsiveness. J. Immunol., 139, 141-147.

Kabilan, L., Troye-Blomberg, M., Patarroyo, M.E. & Perlmann, P. (1987), Regulation of the immune response in P. falciparum. -- IV. T-cell-dependent production of immunoglobulin and anti-Plasmodium falciparum antibodies in vitro. Clin. exp. Immunol., 68, 288-297.

Mond, J.J. & Brunswick, M. (1991), Assays for B cell func- tion, in "Current Protocols in Immuitology", Chap- ter 3, section II, John Wiley & Sons, New York.

Romagnani, S. (1991), Human THI and TH2: doubt no more. lmmunoL Today, 12, 256-257.

Schofield, L. & Uadia, P. (1990), Lack of Ir gene control in the immune response to malaria. J. lmmunol., 144, 2781-2788.

Splawski, J.B., Jenilek, D.F. & Lipsky, P.E. (1989), Im- munomodulatory role of IL4 on the secretion of Ig by human B cells. J. Immunol., 142, 1569-1575.

Van Den Eertwegh, A.J.M., Fasbender, M.J., Schellekens, M.M., Van Oudenaren, A., Boersma, W.J.A. & Claassen, E. (1991), In vivo kinetics and characteri- zation of IFN--t'-producing cells during a thymus- independent immune response. J. lmmunol., 147, 439-446.

Yssei, H., Shanafelt, M.C., Soderberg, C., Schneider, P.V., Anzola, J. & Peltz, G. (1991), Borrelia burg- doferi activates a T helper type-l-like T cell subset in lyme arthritis. J. exp. Med., 174, 593-603.

J. Gysin:

Druiihe and Bouharoun

The assumption that blood stage P. falciparum target epitopes which induce the antibody-dependent

protective immune response are probably not poly- morphic agrees with our recent data showing that passive protection against asexual blood stages can be achieved by transferring monkey or human IgG from "protected" individuals into non-immune falciparum-infected Saimiri monkeys. This suggests that at least some target epitopes are recognized by both hosts and that at least some of them must be cons,:rved and be common to all isolates tested so far. In Aotus monkeys, preliminary results on this mat- ter have been less conclusive, highlighting a possible difference from the Saimiri. This should be of some importance in the interpretation of results related to immunity. Our work indicates that, indeed, monkeys, which are artificial hosts for human malaria can be helpful in malaria-related studies precisely through their amplification of immunological phenomena.

Mattei and Scherf

How likely do autoantibodies developed by the host in response to a P. falciparum infection really play a role in pathology? Also, if indeed a give~ an- tigen induces autoantibodies, this characteristic can vary depending on the species used for immuniza- tion. This introduces difficulties in assessing the im- portance of autoantibodies in malaria infection and immunization.

~'. Zavala:

Comments on Dr. Hommel 's paper

The review by M. Hommel contains some inac- curacies, and I disagree with some of his interpreta- tions and comments.

When referring to vaccine trials in which humans were immunized with P. falciparum sporozoites, he describes the results of Rieckman et al. (WHO Bull. 57 (suppl. 1), 261, 1979) as only partially successful, with 3 out of 8 volunteers being protected. In reali- ty, this study describes two different groups, subject- ed to different schedules of immunization: (a) 3 of the individuals receiving a large immunizing dose, were all protected, and (b) the 4 volunteers receiving a low dose were susceptible to sporozoite-induced malaria. Pooling all the individuals participating in that study in a single group is not only inaccurate but also misleading. Recent humans trials performed in- dependently at the University of Maryland and at the Naval Medical Research Institute fully confirmed the results of the early vaccine trials.

A similar situation is found when the author describes the results of the P. falciparum CS vaccine trials using either synthetic peptides or recombinant

A M A L A R I A D I V E R T I M E N T O "7"r7

protein. These two studies comprise 4 and 5 ex- perimental groups, respectively, which were subjected to different protocols of immunization. Dr. Hom- mel pools all the vaccinees ioto a single group and summarizes the results by stati.ag that only 1/15 and 1/34 were protected. This is inaccurate: in one study, only three volunteers (with detectable circulating an- tibodies), were challenged and 1 was protected. In the other study, only 6 volunteers were challenged and 1 was protected. The subunit vaccines used in both studies were poorly immunogenic, as reflected by the low or even negligible anti-CS antibody titres achieved in all the vaccinees. Still, it is noteworthy that in both trials, the volunteer with the highest an- tibody titre was protected.

Dr. Hommel flatly states that the role of cell- mediated mechanisms was "only investigated after the failure of the NANP vaccines". He seems to be unaware that research on IFN-gamma, T cells and their effects upon the liver stages of malaria were al- ready published or being developed before the reports of the 1987 human trials: Ojo-Amaize (1984), J. tm- munol., 133, 1005; Ferreira et al. (1986), Science, 232, 881 ; Mellouk et al. (1987), J. Immunol . , 139, 4192; Schofield et al. (1987), Nature (Lond.), 330, 664). Further advances in this area could only be achieved using recombinant lymphokines, specific monoclonal antibodies against T-ceU subsets and syn- thetic peptides, reagents which became available only in recent years.

Dr. Hommel overinterprets the negative results on human lymphocyte proliferation after in vitro incu- bation with different synthetic peptides. A negative result on lymphocyte proliferation may indicate the inability to recognize the antigen (restriction) or ab- sence of specific T cells due to poor or non-existent priming. Furthermore, negative results must be in- terpreted with much caution for reasons well ex- plained in the review by Behr and Dubois (this issue) • Lymphocytes may recognize antigen, produce lym- phokines and yet show no in vitro proliferation (Troye-Blomberg et al. (1990), Proc. nat. Acad. Sci. (Wash.), 87, 5484). Clearly, it still remains to be de- termined which is the most adequate parameter to assess antigen recognition by T cells. Consequently, based on negative results, it is not yet possible to predict in vivo T-cell priming after immunization with different epitopes.

Unfortunately, this review does not discuss some fundamental issues regarding the quantitative nature of the protective effect mediated by protective im- mune mechanisms. The capacity of antibodies to pro- tect against parasites depends on their quantity and quality (affinity): these two factors largely determine the formation of antigen-antibody complexes. The amount and quality of the antibodies induced after immunization, will depend on the amount of im- munogen, adjuvants, and molecular structure of the

antigen. It is frequently mentioned (in this review and other publications) that in spite of "high antibody titres" there is poor or no protection. What is a high antibody titre ? Compared to what ? What is the max- imum achievable? Clearly, there is much to learn about all these factors and it is premature, based on a simplistic analysis, to dismiss some antigens as vac- cine candidates.

In the last paragraph of this review, statements supposedly made by anonymous scientists are quot- ed and ridiculed. This is insulting to all scientists in- volved in this area of research. Dr. Hommel should have documented this reference.

General comment

As frequently stated in several papers, studies on the factors involved in the development of naturally acquired immunity may be most important for research aiming at the development of a vaccine. Considering that naturally acquired immunity does not provide sterile immunity, it should not be sur- prising if the most important contribution of these studies is the identification of both antigens not to be used for vaccine development and immune mechanisms not be elicited by the subunit vaccines.

D. Mazier:

V.A. Snewin, S. Longacre and P I4 r},,,,;,4

It is possible to argue equally strongly that P. fai- ciparum is the best adapted of the parasites. It does not commit the "cardinal sin" of causing significant mortality, since it kills less than 1 % of those it in- fects. In biological terms, this is nothing (though medically it ~s a disaster). Linked with this is a high and efficient rate of transmission in the tropics and (perhaps) a greater ability to respond to adverse con- ditions, i.e. introduction of drugs since it is P. fal- ciparum that has developed resistance. I would challenge the implication that P. falciparum by com- parison is a "recent" parasite of humans. They are both well adapted, but in different ways.

The recent findings by Patarroyo et aL that a syn- thetic vaccine based on antigens of P. falciparum will protect those vaccinated against P. vivax as well as P. falciparum does not contradict the established ob- servation that immunity developed naturally does not give cross-protection. The vaccine-induced immuni- ty probably involves epitopes that are "protected" in the native molecule, i.e. are not immunodominant. Nevertheless, when such epitopes are included in ar- tificial induction of an immune response, that im- mune response can be protective.

728 40th FOR UM IN IMMUNOLOG Y

K. Mendis and R. Carter

Even if natural immunity is CD4*-independent, this does not invalidate attempts to vaccinate with peptide Ag, since it is possible to select epitopes that are not naturally the most immunodominant, i.e. beat the parasite at its game of evasion by express- ing only certain dominant T-cell epitopes.

P. Druilhe

Concerning regulatory mechanisms which could take place when the parasite lies inside the hepato- cyte, preliminary results performed using two- dimensional gel electrophoresis and advanced image analysis techniques show that in infected hepatocytes, more than 100 downregulation spots and about 40 upregulation ones appear (I. Humphey-Smith, S. Pied and D. Mazier, unpublished results).

M.E. Perkins:

Commentary on the paper by J.W. Barnwell and M.R. Galinski

Barnwell arid Galinski have given an excellent overview of the complex field of eryP--ocyte- merozoite interactions. The complexity arises from several featu~,~s uf this interaction, particularly the variation both between and within different species of plasmodium. It is now well documented that the principal erythrocyte receptor for P. faMparum is the sialic acid residues of giycophorin, and for P. vivax and P. knowlesi, the receptor is the Duffy glycoprotein. Yet there is ample evidence, at least for P. falciparum and P. knowlesi, that there are alter- nate receptors for some strains of parasites. These so-called alternate or secondary receptors have yet to be identified.

Recently, there have been many reports in the literature on the identification of parasite proteins of P. falciparum, P. knowlesi and P. vivaxthat bind to erythrocytes with the correct specificity expected of receptor-binding proteins. Barnwell and Galinski have questioned the functional significance of such studies. Although the assay used in such studies is indirect, I think it is reasonable to conclude that the proteins identified are receptor-binding proteins. There are some differences between laboratories on the identification of the binding proteins observed, and this may be a result of different labelling condi- tions. However, there are several reasons to support the integrity of the binding assay. Firstly, observa- tions in all laboratories testify to the specificity of this assay. Although numerous (thirty to fifty) pro- teins are added to erythrocytes, generally only one

or two bind strongly; i.e. there is a very low back- ground. Secondly, with a few exceptions, the pro- file of binding to control erythrocytes (i.e. erythrocytes not invaded by the particular plasmodial species) is what would be expected. For instance, the P. vivaxerythrocyte-binding protein of 135-kDa does not bind to Duffy- human erythrocytes. In conclu- sion, I think we can propose that the erythrocyte- binding proteins identified in this assay are function- ing as receptor-binding proteins in the invasion process.

With the introduction of a common binding as- say, it will now be possible to compare some of the parasite-binding proteins identified in different laboratories. It is possible that more than one pro- tein from each species binds to the erythrocyte recep- tor, given the complex make-up of the merozoite surface and apical complex. To date, erythrocyte- binding proteins have been localized to the merozoite surface, apical surface, rhoptry and micronemes. In P. faiciparum, it is interesting to note that while the major merozoite surface protein, MSA-1 binds to hu- man erythrocytes, a rhoptry protein binds to mouse erythrocytes (Sam-Yellowe and Perkins, 1990). This illustrates that a single species of plasmodium is able to use alternate types of proteins for interaction with the host erythrocyte.

Finally, I think it is important in this field to use consistent terminology. Barnwell and Galinski refer correctly to the erythrocyte components with which the parasite interacts as the "erythrocyte receptor". Thus, Duffy glycoprotein and glycophorin are the erythrocyte receptor for P. vivax and P. faMparum, respectively. This is consistent with the terminology used in virology and bacteriology, where the com- ponents on the host cell involved in virus/bacterial recognition are always defined as the receptor. Patho- gen proteins binding to the receptor are defined as receptor-binding proteins. Barnwell and Galinski have referred to the Duffy-binding proteins as Duffy adhesion proteins which is also consistent with this terminology. In contrast, Adams et al., 1990, have referred to the P. knowlesi Duffy-binding proteins as the "Duffy receptor" which is opposite to that normally accepted. Consistent terminology would facilitate our understanding of this complex field.

References

Adams, J.H., Hudson, D.E., Torri, M., Ward, G.E., Wellem, T.E., Aikawa, M. & Miller, L.H. (1990), The Duffy receptor family of Plasmodium knowlesi is lo- cated in the micronemes of invasive malaria merozoites. Cell, 63, 141-153.

Sam-Yellowe, T.Y. & Perkins, M.E. (1990), Binding of Plasmodium falciparum rhoptry proteins to mouse erythrocytes and ther possible role in invasion. Mol. Biochem. Parasit., 39, 91-101).