G. Del Giudice

Tumour necrosis factor-dependent parasite-killing effects during paroxysms in non-immune Plasmodium vivax malaria patients

Plasmodium vivax malaria infections in non‐immune individuals manifest as periodic clinical episodes of fever with chills and rigors known as paroxysms. We have demonstrated that in non‐immune patients the period of paroxysm is associated with the transient presence of plasma factors which kill gametocytes, the intra‐erythrocytic sexual stages of the malaria parasite which transmit the infection from humans to mosquito, rendering them non‐infectious to mosquitoes. Gametocytc killing in paroxysm plasma is mediated by tumour necrosis factor (TNF) acting in conjunction with other essential serum factor(s). Plasma TNF levels were elevated during a paroxysm. In semi‐immune individuals from a P. vivax‐endemic area clinical symptoms of malaria are mild and the parasite killing factors are not induced during paroxysm. Serum TNF levels were correspondingly lower in endemic patients during a paroxysm. Human peripheral blood mononuclear cells (PBMC) can be stimulated in vitro by extracts of P. vivax blood stage parasites to produce TNF and associated parasite killing factor(s), thus simulating in vitro the events that occur during a paroxysm, this being the release of parasite exo‐antigens by rupturing schizonts and the subsequent induction of PBMC to produce TNF and other parasite‐killing factors. We were able to show that convalescent serum from P. vivax semi‐immune individuals block the induction of TNF and parasite‐killing factors by malaria antigens in vitro, presumably through antibodies that neutralize parasite exo‐antigens. Thus, individuals living in malaria‐endemic areas appear to acquire clinical immunity to malaria by avoiding their induction during infection; we have shown that one such mechanism is the neutralization of parasite exo‐antigens that induce the production of parasite killing factors.

Heat shock proteins as carrier molecules: In vivo helper effect mediated by Escherichia coli GroEL and DnaK proteins requires cross-linking with antigen

In the past few years we have shown that mycobacterial heat shock proteins (hsp) of 65 and 70 kD exert a very strong helper effect in mice and monkeys when conjugated to peptides and oligosaccharides and given in the absence of adjuvants. In the present study we show that this adjuvant‐free helper effect (1) is not due to lipopolysaccharide (LPS), since it was observed in LPS‐resistant mice (C3H/HeJ) immunized with hsp‐based constructs containing the malaria peptide (NANP)40, and (ii) is characteristic of hsp, since it was not observed with conjugates containing the mycobacterial p38 antigen, which is not a stress protein. Interestingly, the hsp GroEL and DnaK of Escherichia coli, which share a high degree of homology with the mycobacterial 65‐kD and 70‐kD hsp respectively, exhibit a strong in vivo helper effect when conjugated to the (NANP) peptide and the conjugates given in the absence of adjuvants. This in vivo helper behaviour of the GroEL and DnaK proteins corresponds well to that observed with the mycobacterial 65‐kD and 70‐kD hsp. respectively, since the hsp65‐ and GroEL‐based constructs require previous priming of the animals with live bacille Calmette Guérin (BCG), which is not needed for the hsp70‐ and DnaK‐based constructs. Finally, using both mycobacterial and E. coli hsp we show that their in vivo helper effect in the absence of adjuvants requires cross‐linking to the synthetic peptide. Taken together, our results suggest that the adjuvant‐free helper effect observed with mycobacterial and E. coli hsp may be a generalized phenomenon, exhibited by hsp from diverse microorganisms. These findings may find applications in the design of vaccine constructs.

Specificity of antibodies induced after immunization of mice with the mycobacterial heat shock protein of 65 kD

We have previously shown in mice and monkeys that mycobacterial heat shock proteins (hsp) of 65 and 70 kD exert a strong in vivo helper effect when conjugated to synthetic peptides or bacterial oligosaccharides and given in the absence of any adjuvants. Considering the degree of homology existing in the phylogeny among hsp belonging to the same family, we studied whether antibodies induced in mice with this protocol of immunization with the mycobacterial 65‐kD hsp (hsp65) would cross‐react, and to what extent, with hsp homologues from other origins, notably with the Escherichia coli GroEL protein and with the human homologue (hsp60). The results obtained show that antibodies to the mycobacterial hsp65 cross‐reacted with the E. coli GroEL protein, both in ELISA and Western biol experiments, but not with the human hsp60. In competitive ELISA experiments, the binding of these antibodies to solid‐phase hsp65 was very effectively inhibited by low concentrations of the mycobacterial hsp65; however, for human hsp60. 100 times higher concentrations were required in order to obtain similar patterns of inhibition‐Finally, murine antibodies to the mycobacterial hsp65 always failed to give positive results in Western biol experiments using extracts of murine cells. Taken together, these data suggest that, after immunization of mice with the mycobacterial hsp65 conjugated to peptides or oligosaccharides in the absence of adjuvants, anti‐hsp65 antibodies are induced which cross‐react well with hsp homologues from other prokaryotes (e.g. E. coli GroEL), but which weakly bind the human hsp homologue. These results may have implications for the potential use of microbial hsp molecules in the design of conjugated vaccine constructs.

Hsp70: a carrier molecule with built-in adjuvanticity

One problem associated with the development of subunit vaccines is their limited immunogenicity, due to their physico-chemical structure, their inability to encounter the correct MHC restriction element, or the need for strong adjuvants to be delivered along with them. These problems are usually solved by conjugating target epitopes (peptides or oligosaccharides) with carrier proteins which provide a source of T-cell epitopes recognised by a large proportion of the vaccinated individuals. We have shown that mycobacterial hsp65 and hsp70 exert a strong helper effect in vivo when conjugated to synthetic peptides or oligosaccharides. Interestingly, this helper effect did not require the need for any adjuvant, either in mice or in monkeys. The helper effect mediated by the hsp65 required that animals were previously primed with either live BCG or the hsp65 alone; on the other hand, such a priming was not required when the hsp70 was used in the conjugates. Similar results were obtained with HSP molecules fromEscherichia coli. This may suggest that the adjuvant-free helper effect observed applies not only to mycobacterial HSP, but also to HSP from other prokaryotes. These findings suggest that microbial hsp70 could be considered for the design of conjugated vaccine constructs for eventual human use.

Sporozoite antibodies and malaria in children in a rural area of The Gambia.

Sporozoite antibody levels were measured in a group of children aged one to nine years resident in a rural area of The Gambia, using an ELISA to the repeat peptide (NANP)40. The prevalence and titre of antibodies varied with age but not with sex or ethnic group. Significant variations in prevalence were recorded within a group of adjacent villages. Children who were seropositive at the beginning of the dry season had higher spleen and parasite rates both at this time and at the end of the subsequent rainy season than did seronegative children, suggesting that they were exposed more frequently to infection. However, seropositive children had fewer episodes of fever accompanied by high levels of parasitaemia than did seronegative children, suggesting that they had a greater degree of clinical immunity. No differences were found in seroprevalence rates or in mean antibody titres between children who slept under conventional or Permethrin treated bed nets and those who did not, even though bed nets significantly reduced the number of bites by vector mosquitoes.

Host immune response and pathological expression in malaria: possible implications for malaria vaccines.

Recent progress in parasite immunobiology has led to the identification of several plasmodial antigens representing the target of the protective antibody response of the infected host. As a consequence, some of these antigens have been envisaged as potential malaria vaccines in man. However, in spite of these achievements, the fine mechanisms which lead to the development of a state of partial protective immunity or to the triggering of immunopathology during malaria infection are not yet fully understood. Thus, it may be appropriate to evaluate the relative importance of individual host immune responsiveness to parasite epitopes involved in the induction of immunity, or of some immunologically mediated adverse reactions such as glomerulonephritis, anaemia, thrombocytopenia, and cerebral syndrome.

Can New Vaccines Overcome Parasite Escape Mechanisms

The development of new vaccines remains an essential goal of research dealing with the immunology of infectious diseases. Most existing vaccines are the result of efforts largely based on empiricism. However, in view of the numerous parasite escape mechanisms which characterize many non-vaccine preventable infectious diseases more logical approaches are now required to reach that goal.