Malaria vaccine development: current status.

The development of an effective malaria vaccine represents one of the most important approaches that would provide a cost-effective intervention for addition to currently available malaria control strategies. Here, Howard Engers and Tore Godal review recent advances. Over the past decade there has been considerable progress in the understanding of immune mechanisms involved in conferring protection to malaria and in the identification of vaccine candidate antigens and their genes. Several new vaccines have entered Phase I/II trials recently, new adjuvants have been developed for human use and new approaches, such as DNA vaccines and structural modification of antigens to circumvent some of the strategies the parasite uses to avoid the immune response, are being applied. Thus, from the TDR perspective, global malaria vaccine development is entering a crucial period with unprecedented opportunities.

Progress on vaccines against parasites.

There has been significant progress in attempts to develop effective vaccines against parasitic diseases, including malaria, leishmaniasis and schistosomiasis. In malaria, in addition to field trials with SPf66, the Colombian malaria vaccine, several Plasmodium falciparum candidate vaccines are under Phase I testing, including NYVAC-7, a multi-antigen, attenuated recombinant vaccinia virus. Additional candidate antigens are at an advanced stage of pre-clinical development. In leishmaniasis, Phase III clinical trials on first generation vaccines (killed Leishmania, with or without BCG) are proceeding in several countries. Use of IL-12 as an adjuvant for use with killed Leishmania vaccine is being studied in non-human primates. A genetically constructed (gene knock-out) live avirulent Leishmania is being developed in preclinical studies as a potential live vaccine. Research is also underway to evaluate several recombinant proteins. The genes coding for such leading candidate antigens are also being incorporated into various live vectors to yield recombinant organisms with vaccination potential. In schistosomiasis, a strategy for the development of a vaccine against Schistosoma mansoni has been established, focussing on six priority recombinant antigens. An Asian S. japonicum vaccine development network has also been established, initially to develop a vaccine to block transmission in cattle and oxen, important reservoirs of the disease in Asia, and ultimately a human vaccine. As all of the above-mentioned vaccines will be used in the field in disease-endemic tropical countries, optimal stability will be of paramount importance.

Immunogenicity of a non-repetitive sequence of Plasmodium falciparum circumsporozoite protein in man and mice.

In the present work, the hypothesis that individuals naturally exposed to Plasmodium falciparum malaria infection in endemic areas produce antibodies directed against non-repetitive epitopes of the circumsporozoite protein was investigated. Using a synthetic peptide reproducing the non-repetitive group-conserved region I sequence, we have shown that specific anti-region I antibodies are detectable in sera from endemic countries. Of these sera, 87% also had antibodies against the immunodominant repetitive epitope (Asn-Ala-Asn-Pro, NANP) of P. falciparum. In order to study the immunogenicity of this non-repetitive epitope, a synthetic peptide consisting of both region I and three (NANP) repeats [RI-(NANP)3] was used to immunize inbred strains of mice. H-2b mice produced antibodies against both the repetitive and the non-repetitive epitope. These antibodies were specific for each epitope, recognized P. falciparum sporozoites in immunofluorescence, and inhibited sporozoite penetration into human liver cells in vitro. Non-H-2b mice were completely unresponsive. Lymph node cells from H-2b mice immunized with RI-(NANP)3 peptide proliferated in the presence of RI-(NANP)3 and of (NANP)4 peptide, but never in the presence of RI peptide alone. These findings demonstrate that in the configuration used (i) the non-repetitive epitope region I does not carry T-helper epitopes; (ii) the (NANP) repetitive epitope may act as a carrier for the immune response to region I in mice; and (iii) therefore, immune response to region I in man probably depends on the recognition of T-cell epitopes similar to those involved in the anti-NANP response: i.e. such a T epitope may be NANP itself in responding individuals or another, not yet recognized, sporozoite T-cell epitope.

Antibodies to the repetitive epitope of Plasmodium falciparum circumsporozoite protein in a rural Tanzanian community: a longitudinal study of 132 children.

An ELISA employing a novel synthetic peptide consisting of 40 (Asn-Ala-Asn-Pro) repeats of Plasmodium falciparum circumsporozoite protein, (NANP)40, was used to detect antibodies against P. falciparum circumsporozoite protein in 132 children, 1 month to 15 years old, from a rural community (Kikwawila village) of Tanzania, a region where malaria is hyperendemic. The children were surveyed comprehensively over 3 consecutive years for clinical, parasitological, and serological parameters. Entomological data were also gathered for selected households in this village. The following results were obtained: anti-(NANP)40 antibodies increased as a function of age; the majority of children over 10 years showed a stable positivity for such antibodies during the longitudinal study; a negative correlation was observed between the levels of anti-sporozoite antibodies and both spleen enlargement and the presence of parasites in thick smears; no relationship was found between anti-(NANP)40 antibodies and asexual blood stage antibodies; children living in two representative households with comparable indoor resting mosquito densities showed markedly different frequencies of anti-(NANP)40 antibodies, in spite of comparable clinical, parasitological, and serological parameters. Thus, in addition to the exposure to infectious mosquito bites, other (e.g., genetic) factors, may play a role in the ability of certain individuals to mount an antibody response against this immunodominant repetitive epitope. The results presented in this paper confirm that the (NANP)40-ELISA represents a simple, reliable means for the detection of anti-(NANP)40 circumsporozoite protein antibodies and suggest that such antibodies may contribute to the immune protection against malaria in humans.

Functional activity in vivo of effector T cell populations. III. Protection against Moloney murine sarcoma virus (M-MSV)-induced tumors in T cell deficient mice by the adoptive transfer of a M-MSV-specific cytolytic T lymphocyte clone.

The functional activity of Moloney murine sarcoma virus (M-MSV)-specific T lymphocytes in vivo was assayed by the i.v. injection of virus-specific T lymphocytes into T cell-deficient "B mice". Virus-specific T lymphocytes generated in mixed lymphocyte tumor cell cultures were transferred i.v. into syngeneic "B mice" injected simultaneously at a distant site with the virus. These experiments indicated that a low dose (1 X 10(6) cultured cells) of infused lymphocytes can afford protection. To define the T lymphocyte subpopulation which was active, Lyt-2+ lymphocytes were selected by "panning" on plastic petri dishes coated with anti-Lyt-2 monoclonal antibody, and Lyt-2- lymphocytes selected by treatment with anti-Lyt-2 monoclonal antibody and complement. The results indicated that a Lyt-2+ lymphocyte-enriched population was more efficient in conferring protection against M-MSV-induced tumors. To investigate if cytolytic T lymphocytes (CTL) alone had a protective effect, a M-MSV-specific CTL clone was transferred in the same model system. The results demonstrated that a M-MSV-specific CTL clone prevented M-MSV-induced tumor growth and also induced the destruction of syngeneic Moloney murine leukemia virus (M-MuLV)-induced MBL-2 leukemic cells in the peritoneal cavity. However, the cell dose required to obtain protection using a CTL clone was higher than that which was effective when mixed lymphocyte tumor cell culture cells were used. To assess the ability of the transferred cells to home and to repopulate the lymphoid organs of the "B mice", the frequency of virus-specific CTL precursors in the spleen was evaluated by limiting dilution analysis. The results indicated that lymphocytes from mixed lymphocyte tumor cell cultures can be recovered from the spleens of "B mice" injected i.v. 25 days earlier. On the contrary, following the transfer of an active CTL clone, a very low frequency (less than 1/200,000 cells) of virus-specific CTL precursors was present in the spleens of recipient animals. The same M-MSV-specific CTL clone did not yield protection against M-MSV-induced tumors or MBL-2 leukemic cells when injected i.v. into M-MuLV tolerant mice.

Detection of human antibodies against Plasmodium falciparum sporozoites using synthetic peptides.

A large peptide consisting of about 40 (Asn-Ala-Asn-Pro) repeats of Plasmodium falciparum circumsporozoite protein, (NANP)40, was synthesized. It was recognized specifically by monoclonal antibodies produced against P. falciparum sporozoites. Moreover, this peptide strongly inhibited the binding of such monoclonal antibodies to antigens present in a sporozoite extract. The (NANP)40 peptide was employed without any carrier to develop an enzyme-linked immunosorbent assay to detect sporozoite-specific serum antibodies arising after natural malaria infections. Antibodies were detected in a high percentage (43.1%) of European patients suffering from acute P. falciparum malaria and in Africans living in an area of Gabon endemic for malaria. In the latter group, the frequency of antisporozoite antibodies increased with age, reaching 65.9% in individuals more than 40 years old. There was a significant correlation between the results obtained with an immunofluorescence assay with glutaraldehyde-fixed sporozoites and those obtained by enzyme-linked immunosorbent assay with (NANP)40. Therefore, such synthetic peptides representing the repetitive epitope of P. falciparum circumsporozoite protein can be used for the detection of antisporozoite antibodies and for the epidemiological studies required to obtain base-line data concerning the immune status of individuals before their participation in a sporozoite vaccine trial.

Cells of T-lymphocyte origin produce an erythropoietin-like growth factor.

Erythropoietin-like activity (EpLA) is one of several hematopoietic growth factors found in spleen cell-conditioned medium. Conditioned media obtained from both dissociated tissues and cloned cell lines were examined in order to define the cellular source of EpLA. The growth factor was only found in supernatants of tissues that contained T-lymphocytes. An examination of media conditioned by cloned cell lines representative of the major cell types in spleen (T- and B-lymphocytes, macrophages) confirmed the association between T cells and EpLA production. Limiting dilution analyses established that normal T-lymphocyte clones produced EpLA, but this property was not correlated with a specific functional T-cell subset. On stimulation with PMA, the T-cell lymphoma cell line, EL-4, proved to be the most potent producer of EpLA found to date.

The antibody response in mice to carrier-free synthetic polymers of Plasmodium falciparum circumsporozoite repetitive epitope is I-Ab-restricted: possible implications for malaria vaccines.

The immunogenicity of a novel synthetic peptide consisting of an average of 40 (Asn-Ala-Asn-Pro) repeats of the circumsporozoite protein of Plasmodium falciparum, (NANP)40, was studied in mice without using any carrier proteins. First, high titers of anti-(NANP)40 antibodies could be obtained after immunization of C57BL/6 mice. These antibodies also reacted with an extract of mosquitoes infected with P. falciparum sporozoites. C57BL/6 nu/nu mice did not produce antibodies against (NANP)40. Secondly, when 14 strains of mice with nine different H-2 haplotypes were immunized with (NANP)40 without carrier, only H-2b mice were found to produce anti-(NANP)40 antibodies, whereas all non-H-2b mice were consistently unresponsive. This response was demonstrated to be I-A-linked by using recombinant and mutant mice. I-Ab [B10.A(5R)] mice produced anti-(NANP)40 antibodies as well as H-2b inbred mice. B6CH-2bm12 I-Ab-mutant mice showed only a very low response. Third, the antibody response against (NANP)40 could be induced in nonresponder mice by immunization with the peptide coupled to a carrier protein. In view of the existence of such an exceptional H-2b restriction in the response to sporozoite synthetic peptides in mice, the triggering of peptide-specific T cell responses in humans receiving sporozoite malaria vaccines might be difficult to achieve.

Synthetic Plasmodium falciparum circumsporozoite peptides elicit heterogenous L3T4+ T cell proliferative responses in H-2b mice.

The ability of synthetic P. falciparum (NANP)n circumsporozoite peptides to elicit murine T cell proliferative responses was studied. When C57BL/6, C3H, and DBA/2 mice were injected with (NANP)40, only C57BL/6 (H-2b)-immune lymph node cells proliferated on restimulation in vitro with the same peptide. By using anti-I-A monoclonal antibodies or spleen cells from congenic H-2b mice as a source of antigen-presenting cells, the T cell proliferative response was shown to be restricted to the I-Ab region of the C57BL/6 haplotype. These results are in agreement with previous experiments which demonstrated that the anti-(NANP)40 antibody response was uniquely restricted to C57BL/6 (H-2b) mice. Several C57BL/6 long-term (NANP)n-specific T cell lines and clones were derived. All of the clones exhibited the L3T4 helper T cell phenotype. A considerable heterogeneity of T cell responses was observed when the lines and clones were stimulated with different concentrations of the various peptides studied. The results, together with the observed genetic restriction for both antibody and T cell responses, suggest that perhaps not all individuals who receive a similar repetitive tetrapeptide sporozoite malaria vaccine will develop T cell and or antibody responses.

L3T4+ T lymphocytes play a major role in the pathogenesis of murine cerebral malaria.

The pathogenic importance of L3T4+ T cells in the development of murine cerebral malaria was demonstrated by the following observations. First, in vivo administration of an anti-L3T4 monoclonal antibody protected Plasmodium berghei-infected CBA mice from the development of neurologic symptoms and acute death. In contrast, injection with an MAb directed against the Ly.2+ T cell subset had no protective effect. Second, thymectomized, irradiated, and bone marrow reconstituted (ATxBM) CBA mice did not develop acute cerebral malaria when infected by P. berghei, although parasitemia and anemia rose to the same extent as in normal P. berghei-infected CBA mice. The occurrence of lethal neurologic perturbations could be restored in ATxBM mice selectively reconstituted with L3T4+ Ly.2-lymphocytes but not with Ly.2+ L3T4- cells. Third, adoptive transfer of L3T4+ cells from mice dying of cerebral malaria into euthymic mice subsequently infected by P. berghei led to an acceleration of the disease. These results confirm that cerebral malaria in mice is the expression of immunopathologic reactions and outline the particular pathogenic importance of L3T4+ T cells.

Detection of antibodies to Plasmodium falciparum sporozoites by employing synthetic peptides.

A new achievement in the immunodiagnosis of malaria has been reached after the knowledge of the molecular structure of some plasmodial antigens has become available. One example is given by the repetitive immunodominant epitope of Plasmodium falciparum circumsporozoite protein, which consists of 4 tandemly repeated aminoacids (Asn-Ala-Asn-Pro = NANP). A large synthetic peptide reproducing 40 NANP repeats, (NANP)40, has been shown to reproduce efficiently the native antigen in the CS protein and has been used to develop an enzyme-linked immunosorbent assay (ELISA) for the detection of antisporozoite antibodies in individuals from malaria-endemic countries. This (NANP)40 ELISA has been employed in a longitudinal study in a rural community in Tanzania. The results obtained have shown (i) that the presence of anti-(NANP) antibodies is associated with a certain degree of protective immunity; and (ii) that genetic factors could play a role in the host immune responsiveness to (NANP). Such an ELISA can be easily applied to field research and can be useful for monitoring the immune status of populations participating, in the future, to malaria vaccination trials employing P. falciparum sporozoite peptides.

Pathogenicity of fibroblast- and lymphocyte-specific variants of minute virus of mice.

We tested two strains of the minute virus of mice (MVM) for pathogenic effects and patterns of infection in laboratory mice. The two strains differ in their ability to infect differentiated cultured cells: the prototype virus, MVMp, infects only fibroblasts, while its variant, MVMi, is restricted to lymphocytes. We find that neither strain has any demonstrable effects on the T-cell function of mice infected as adults. In contrast, MVMi, but not MVMp, is able to induce a runting syndrome accompanied by mild immune deficiencies upon the infection of newborn mice. After neonatal infection, MVMi spreads to many organs, and the presence of viral replicative form DNA is evident in nucleic acid hybridization experiments. In contrast, replication of MVMp can be detected only by the seroconversion of infected animals. Newborn mice that grow abnormally as a result of MVMi infection also have low circulating antibody titers to the virus. This phenomenon may be a consequence of the lymphotropism of MVMi.

Characterization of gP85gag as an antigen recognized by Moloney leukemia virus-specific cytolytic T cell clones that function in vivo.

The gag membrane protein gP85gag, encoded by Moloney murine leukemia virus (M-MLV), was identified as a target molecule recognized by Moloney murine sarcoma virus--M-MLV (M-MSV--M-MLV)-specific cytolytic T lymphocyte (CTL) clones. Target cells infected with Ab-X-MLV, an M-MLV-derived mutant virus not encoding gP85gag, were not lysed by the CTL clones. The same CTL clones were shown previously to induce the destruction of M-MLV-induced tumor cells in the peritoneal cavity. We have now characterized CTL-resistant antigen-loss tumor cell variants that have lost the surface antigen, but which retain transcriptionally silent M-MLV genomes. A cloned antigen-loss variant that reverted in vitro to the CTL-susceptible phenotype reexpressed M-MLV genomes that had undergone an insertion event in the region of the viral DNA coding for the gag membrane protein. Intravenous injection of virus-specific CTL clones inhibited tumor formation in mice injected subcutaneously with M-MSV--M-MLV.

Monoclonal antibodies against mycobacterial antigens.

The World Health Organization (WHO) scientific working groups on the Immunology of Leprosy (IMMLEP) and Tuberculosis (IMMTUB) recently organized two international workshops designed to characterize the specificity and reaction patterns of approximately 55 murine monoclonal antibodies (mAbs) which had been raised against Mycobacterium leprae or Mycobacterium tuberculosis and submitted to the IMMLEP and IMMTUB monoclonal antibody banks.

Differential effects of two anti-apo-cytochrome c-specific monoclonal antibodies on the function of apo-cytochrome c-specific murine T cell clones.

A murine monoclonal antibody (SJL 2-4) specific for the antigen apo-cytochrome c was shown to inhibit both antigen-induced proliferation and lymphokine secretion by an apo-cytochrome c-specific BALB/c helper T cell clone. The inhibition was specific because additional apo-cytochrome c-specific T cell clones were not inhibited by the same monoclonal antibody. Time course studies of the inhibition indicated that the initial 8 hr of contact between T cell clones and antigen-presenting cells were critical for activation of the T cell clones. Inhibition of T cell functions by antigen-specific antibodies appeared to correlate with the antibody-antigen binding constant because a second monoclonal antibody (Cyt-1-59), with identical specificity but with a lower affinity constant for apo-cytochrome c, had very little inhibitory effect on the proliferation or lymphokine secretion of apo-cytochrome c-specific T cell clones.

Exacerbation of murine cutaneous leishmaniasis by adoptive transfer of parasite-specific helper T cell populations capable of mediating Leishmania major-specific delayed-type hypersensitivity.

The effect of adoptive transfer of in vitro-propagated Leishmania major-specific T cell populations on the course of experimentally induced cutaneous leishmaniasis was studied in mice. The L. major-specific T cells expressed the T helper/inducer phenotype and were able in vitro to a) mount a specific proliferative response, b) provide specific helper activity for antibody responses, c) activate parasitized macrophages resulting in L. major destruction, and d) secrete macrophage-activating factors as tested in a tumoricidal assay. These T cells were also found capable of transferring parasite-specific delayed-type hypersensitivity responses to normal syngeneic mice. Results indicated that the i.v. transfer of these L. major-specific T cell populations into normal syngeneic mice exacerbated cutaneous lesions induced by infection with L. major. This effect on the disease process appeared to be dependent upon recognition of parasite antigens by the injected T cells because no exacerbation of the disease process was seen after the transfer of similar T cell populations specific for an antigen unrelated to the parasite, namely ovalbumin. However, the inclusion of ovalbumin in the L. major infecting inoculum resulted in an exacerbating effect of ovalbumin-specific T cells on cutaneous leishmaniasis. These unexpected results were supported by observations showing that immunization of mice with L. major antigens in complete Freund's adjuvant 7 days before infection with L. major led to exacerbated lesions. A similar aggravation of L. major-induced cutaneous lesions was also observed in mice previously immunized with an unrelated antigen provided that this antigen was included in the L. major infecting inoculum.