Use of monoclonal antibodies to detect antigens of malaria parasites
Use of infection sera to detect antigens of malaria parasites
Identification of protective antigens of Anaplasma organisms
I.F. Zavala
Rapporteur's summary
There are three possible targets for a malaria vaccine, i.e. the sporozoites, merozoites and gametes. Previous work has demonstrated the possible use of sporozoites as the immunogen. For example, Mulligan in the 1940s used UV-irradiated sporozoites when working on avian malaria. Nussenzweig working with Plasmodium berghei in rodents and P knowlesi and P cynomolgi in monkeys used X or gamma-irradiated sporozoites, while Clyde and McCarthy, working with P falciparum and P vivax in humans, used irradiated sporozoites.
Antibodies to the protective antigens on the sporozoite surface produce a capping-like phenomenon called the circumsporozoite precipitation (CSP) reaction, i.e. an antibody-mediated shedding of the sporozoite surface coat. However, it is not possible to remove all the CS protein molecules from the parasite surface by this process.
Monoclonal antibodies and hyperimmune serum raised against P berghei sporozoites both recognized a 44,000-dalton protein which occurs on the parasite surface. Blocking studies using Fab fragments suggested that the sporozoite molecule (CS protein) may be involved in hepatocyte invasion.
A family of sporozoite surface antigens is present in four different Plasmodium species. These antigens differ slightly in molecular mass (approximately 44,000 daltons) and isoelectric points. Higher molecular mass precursors of these proteins have also been identified.
The CS protein has one immunodominant region as assessed by competition and inhibition of polyclonal antibody binding using monoclonal antibodies. The sporozoite surface antigens of P vivax, P falciparum and P knowlesi all have repeated epitopes in the immunodominant region, and between species the repeated epitope differs in the total number of amino acids, specific amino acids and number of repeats. Other areas of the sporozoite antigen (flanking regions) are more uniform and there is some similarity between the different species.
In P falciparum the epitope must contain at least three repeats before an effective antibody response is elicited. Similarly human hyperimmune serum appears to require a three-repeat epitope for recognition.
Immunization with a synthetic three-epitope repeat of P falciparum coupled to tetanus toxoid produces high antibody titres in a dose-dependent manner. Without the addition of adjuvant, antibody of a lower titre was elicited. Most antibodies to the synthetic peptide recognize sporozoites in the immunofluorescence antibody test (IFAT) and homologous sporozoites inhibited binding of antibody to the peptide immunogen but heterologous sporozoites did not. Antibodies to synthetic peptides inhibited hepatocyte invasion by P falciparum sporozoites.
The choice of suitable carriers and adjuvants needs careful consideration before synthetic antigens can be used as a vaccine against malaria in man.
Points arising from the discussion
No antigens other than the CS protein could be detected on the surface of the sporozoite even after antibody-induced shedding of most of the surface coat.
In terms of antigenicity, synthetic sporozoite antigens containing three to five repeated epitopes were equally immunogenic.
When synthetic peptides were Used to immunize monkeys against P knowlesi infections, only partial protection was achieved. However, sera from immunized monkeys contained sporozoite-neutralizing antibodies. The incubation of sporozoites in such sera before infection of monkeys abolished their infective capacity.
Monoclonal antibodies to sporozoite antigens did not react with any subsequent stage of the parasite lifecycle. However, antibodies to the merozoite stages recognized sporozoite antigens.
Antibodies may act by inhibiting sporozoite binding to liver cells. It has been proposed that the CS protein recognizes a receptor on the hepatocytes. The antibodies alone do not always cause irreversible damage to the sporozoite. The role of cell-mediated reactions in protection against the sporozoite form of the parasite is unknown, but, because of the short time (30 min) the sporozoites take to reach the liver cells, it is difficult to envisage a major role for cell-mediated immunity at this stage.
In malaria endemic areas, children under 10 years old have very low anti-sporozoite antibody titres whereas adults over 25 have antibodies in their serum which recognize the immunogenic peptide fragment of the sporozoite surface antigen. Trials of the synthetic peptide vaccine in man are being planned as are further trials in Aotus monkeys, although the variability of infections in these monkeys limits their use as a good model for human malaria.
G.V. Brown
Rapporteur's summary
Patients in malaria endemic areas who, in the face of continuing high challenge, show only occasional parasitaemia and minimal clinical symptoms can be considered to be immune. Humoral immunity is known to be important since serum from immune patients has been used to treat children with acute disease. Growth of in vitro-propagated Plasmodium falciparum is inhibited by immune serum (although large amounts may be needed). Human serum from endemic areas can be used to screen for P falciparum antigens that are protective.
Relevant molecules of parasite origin can be selected by analysis of human sera using the following approaches:
(1) checking the rise of antibody titres against specific proteins in semi-immune individuals during convalescence(2) longitudinal studies of antibody specificity as individuals acquire immunity
(3) studying the effect of immune serum on in vitro growth of the parasite and comparing the specificities of inhibitory versus noninhibitory immunoglobutins
(4) for strain-specific antigens, neglecting all clones recognized by antiserum (rabbit or human) against a closely related strain and selecting those preferentially recognized by the serum of interest.
When immunoprecipitated, biosynthetically labeled P falciparum extracts were analysed by SDS-PAGE electrophoresis, sera of exposed individuals (immune and nonimmune) recognized many proteins. No single antigen was found that correlated completely with the development of immunity. However, a negative correlation was noted with a 96, 000-dalton protein which was never recognized by sera from acutely ill patients; the antibody which inhibited in vitro growth recognized this protein.
Significant advances have been made in the identification of relevant parasite antigens by the use of parasite expression libraries. These libraries were probed with sera from both acute and convalescent patients. Several fusion proteins were recognized differentially by the different sera, e.g. inhibitory compared with noninhibitory immunoglobulins or serum having antibody to strain-specific S-antigen.
Bacterial clones containing expressed antigens could be injected directly into mice to produce antisera to cloned proteins (for immunoprecipitation, IFAT etc). Affinity columns using the bacterially synthesized parasite fusion proteins allowed the purification of monospecific, polyclonal antibodies from sera. These antibodies were then used to probe Western blots of proteins from different lifecycle stages of the parasite. The sera were also used in IFAT to localize stage-specific parasite antigens.
One particular protein was identified in all asexual stages. This protein was sequenced and a portion was found that was similar in sequence to the circumsporozoite protein. This is termed the 'circumsporozoite-related antigen'.
When different isolates of P falciparum were examined, heterogeneity in molecular mass of some proteins was apparent. The molecular masses of other antigens did not vary between different isolates. Other proteins, such as S-antigens, were isolate-specific.
One protein frequently recognized by convalescent sera was specific for the schizont stage of the parasite. A protein was found on the surface of red blood cells infected with ring stages (and other stages). It possessed two regions of repeated sequences, one near the 5' end and another at the 3' end. Different individuals showed variable antibody responses to the two areas of repeated sequences. Purified fusion proteins are used as substrates in enzyme-linked immunosorbent assay (ELISA) testing for seroepidemiology using defined antigens.
There are several constraints on the analysis of human immune response: many antigenic specificities could be important in protection, many protective mechanisms may be involved, in vitro studies do not necessarily reflect the in vivo situation, and the immune response at the level of individual antigenic determinants must be elucidated (cloned antigens can now be used in cell-mediated immunoassays and seroepidemiology).
Points arising from the discussion
The class and subclass of human antibodies to stage-specific parasite antigens have yet to be determined. A high proportion of human sera from endemic areas have anti-merozoite antibodies and individuals with long exposure may have anti-sporozoite antibodies. The epitope recognized by the anti-merozoite sera on the sporozoite has not yet been determined.
G. Palmer
Rapporteur's summary
Anaplasmosis is an infectious arthropod-borne disease of cattle. The infectious agent, Anaplasma marginale, is an abnormal rickettsia transmitted through ticks, mechanical vectors (flies and ticks) or blood-contaminated fomites.
The disease has an incubation period of 3 to 8 weeks, a development period of 4 to 10 days, and if death does not occur, a period of convalescence with the majority of eases producing a carrier state. Experimentally up to 95% parasitaemia of the red blood cells can occur and 10% mortality can occur at crisis, the point of maximum packed red cell volume (PCV) depression. Parasite initial bodies initiate infection by invading red cells where they then replicate.
Rabbits were immunized with purified initial bodies to show that neutralizing antibodies could be induced. The resultant antiserum when preincubated with A marginale protected splenectomized cattle from infection by antibody-treated initial bodies.
Immunoprecipitation revealed five parasite surface antigens which showed no cross-reactivity with red blood cell surface antigens. Two monoclonal antibodies recognizing epitopes on antigen Am 105, when mixed, completely neutralized the infectivity of 107 initial bodies (but not higher numbers). Extension of the prepatent period occurred in splenectomized cattle even though infection was established. Competition between these monoclonal antibodies showed reciprocal inhibition. The antigen is protease sensitive and all tests for carbohydrate content have so far proved negative. While different geographical isolates can be distinguished on the basis of the recognition pattern of other monoclonal antibodies, all carry the antigen Am 105 which is reactive with neutralizing monoclonal antibodies.
Purified, detergent-solubilized initial bodies and monoclonal antibodies in affinity columns were used to purify Am 105. Antigen was eluted from the immunoabsorbent column with 0.5% deoxycholate and 2 M KSCN in Tris buffer. DOC was chosen as the detergent as it is easily removed by dialysis whereas NP40 is not. The column can be regenerated after this elusion procedure.
Cattle were given four doses at 100 micrograms of antigen in complete Freund's adjuvant per dose at 14-day intervals. Antibody titres estimated by ELISA against the antigen were 104 to 105. On challenge, 60% of the cattle showed infection but with low parasitaemia and an extended prepatent period; 40% of the cattle did not show parasitaemia. Those cattle not showing parasites were still carriers as their blood generated infections when subinoculated into splenectomized cattle
Points arising from the discussion
The mode of action of the protective antibodies is uncertain. The relevant parasite antigens have been shown to be present on the parasite surface but not on the surface of the infected red blood cells.
Immunized cattle were challenged by inoculation with purified parasites rather than infected ticks. It is not yet clear why naturally infected cattle do not produce detectable neutralizing antibodies. A 36,000-dalton parasite antigen also proved to be partially protective.
