J. A. M. Langermans

Divergent effect of bacillus Calmette–Guérin (BCG) vaccination on Mycobacterium tuberculosis infection in highly related macaque species: Implications for primate models in tuberculosis vaccine research

Despite the widespread use of bacillus Calmette–Guérin vaccination, Mycobacterium tuberculosis infection remains globally the leading cause of death from a single infectious disease. The complicated and often protracted dynamics of infection and disease make clinical trials to test new tuberculosis vaccines extremely complex. Preclinical selection of only the most promising candidates is therefore essential. Because macaque monkeys develop a disease very similar to humans, they have potential to provide important information in addition to small animal models. To assess the relative merits of rhesus and cynomolgus monkeys as screens for tuberculosis vaccines, we compared the efficacy of bacillus Calmette–Guérin vaccination and the course of infection in both species. Unvaccinated rhesus and cynomolgus monkeys both developed progressive disease with high levels of C-reactive protein, M. tuberculosis-specific IgG, and extensive pathology including cavitation and caseous necrosis. Bacillus Calmette–Guérin vaccination protected cynomolgus almost completely toward the development of pathology, reflected in a striking 2-log reduction in viable bacteria in the lungs compared with nonvaccinated animals. Rhesus, on the other hand, were not protected efficiently by the bacillus Calmette–Guérin. The vaccinated animals developed substantial pathology and had negligible reductions of colony-forming units in the lungs. Comparative studies in these closely related species are likely to provide insight into mechanisms involved in protection against tuberculosis.

Antibodies to Glycans Dominate the Host Response to Schistosome Larvae and Eggs: Is Their Role Protective or Subversive?

Multiple exposures of chimpanzees to the radiation-attenuated schistosome vaccine provoked a strong parasite-specific cellular and humoral immune response. Specific IgM and IgG were directed mainly against glycans on antigens released by cercariae; these were also cross-reactive with soluble antigens from larvae, adult worms, and eggs. Egg deposition was the major antigenic stimulus after challenge of vaccinated and control chimpanzees with normal parasites, eliciting strong antiglycan responses to egg secretions. Glycan epitopes recognized included LacdiNAc, fucosylated LacdiNAc, Lewis(X) (weakly), and those on keyhole limpet hemocyanin. Antibodies to peptide epitopes became prominent only during the chronic phase of infection, as glycan-specific IgM and IgG decreased. Because of their intensity and cross-reactivity, the antiglycan responses resulting from infection could be a smoke screen to subvert the immune system away from more vulnerable larval peptide epitopes. Their occurrence in humans might explain the long time required for antischistosome immunity to build up after infection.

Cellular and humoral immune responses and protection against schistosomes induced by a radiation-attenuated vaccine in chimpanzees.

ABSTRACT The radiation-attenuated Schistosoma mansoni vaccine is highly effective in rodents and primates but has never been tested in humans, primarily for safety reasons. To strengthen its status as a paradigm for a human recombinant antigen vaccine, we have undertaken a small-scale vaccination and challenge experiment in chimpanzees (Pan troglodytes). Immunological, clinical, and parasitological parameters were measured in three animals after multiple vaccinations, together with three controls, during the acute and chronic stages of challenge infection up to chemotherapeutic cure. Vaccination induced a strong in vitro proliferative response and early gamma interferon production, but type 2 cytokines were dominant by the time of challenge. The controls showed little response to challenge infection before the acute stage of the disease, initiated by egg deposition. In contrast, the responses of vaccinated animals were muted throughout the challenge period. Vaccination also induced parasite-specific immunoglobulin M (IgM) and IgG, which reached high levels at the time of challenge, while in control animals levels did not rise markedly before egg deposition. The protective effects of vaccination were manifested as an amelioration of acute disease and overall morbidity, revealed by differences in gamma-glutamyl transferase level, leukocytosis, eosinophilia, and hematocrit. Moreover, vaccinated chimpanzees had a 46% lower level of circulating cathodic antigen and a 38% reduction in fecal egg output, compared to controls, during the chronic phase of infection.

A prime-boost immunisation regimen using DNA followed by recombinant modified vaccinia virus Ankara induces strong cellular immune responses against the Plasmodium falciparum TRAP antigen in chimpanzees

Two chimpanzees were vaccinated intramuscularly against malaria using plasmid DNA expressing the pre-erythrocytic antigens thrombospondin related adhesion protein (PfTRAP) and liver stage specific antigen-1 (PfLSA-1) of Plasmodium falciparum together with GM-CSF protein. A recombinant modified vaccinia virus Ankara (MVA) expressing PfTRAP was injected intramuscularly 6 weeks later to boost the immune response. This sequence of antigen delivery induced a specific and long-lasting T cell and antibody response to PfTRAP as detected by ELISPOT assay and ELISA. Antibody responses were detected after four DNA injections, and were boosted by injection of recombinant MVA expressing PfTRAP. Interferon-gamma secreting antigen-specific T cells were detected in both animals, but only after boosting with recombinant MVA. By screening a panel of PfTRAP-derived peptides, an epitope was identified that was recognized by cytotoxic T lymphocytes in one of the chimpanzees studied. T cells specific for this epitope were present in PBMCs and liver-infiltrating lymphocytes at a frequency of between 1 in 200 and 1 in 500. The high immunogenicity of this prime-boost regimen in chimpanzees supports further assessment of this delivery strategy for the induction of protection against P. falciparum malaria in humans.

Effect of lisofylline and pentoxifylline on the bacterial-stimulated production of TNF-alpha, IL-1beta and IL-10 by human leucocytes

The present study concerns the effect of the xanthine derivates lisofylline (LSF) and pentoxifylline (PTX) on the production of pro‐inflammatory cytokines tumour‐necrosis factor‐α (TNF‐α) and interleukin‐1β (IL‐1β) and the de‐activating cytokine interleukin‐10 (IL‐10) by human leucocytes during stimulation with lipopolysaccharide (LPS), heat‐killed Gram‐negative bacteria (GNB) or Gram‐positive bacteria (GPB). The production of TNF‐α and IL‐1β by leucocytes stimulated with LPS, Haemophilus influenzae type b (Hib) or Streptococcus pneumoniae was inhibited by both drugs. The production of IL‐10 by leucocytes stimulated with LPS and Hib was inhibited by both xanthine derivates only at 48u2003hr. However, incubation of leucocytes with S. pneumoniae in the presence of LSF or PTX stimulated the production of IL‐10 about four‐ and twofold at 24u2003hr and 48u2003hr, respectively. In all instances, the extent of inhibition or enhancement of cytokine production by LSF or PTX was equal. The divergent effects of xanthine derivates on the IL‐10 production indicate the existence of distinct intracellular pathways depending on whether leucocytes are stimulated by GPB or GNB.

Elimination of Schistosoma mansoni Adult Worms by Rhesus Macaques: Basis for a Therapeutic Vaccine?

BACKGROUNDnAmong animal models of schistosomiasis, the rhesus macaque is unique in that an infection establishes but egg excretion rapidly diminishes, potentially due to loss of adult worms from the portal system via shunts or death by immune attack.nnnPRINCIPAL FINDINGSnTo investigate this, six rhesus macaques were exposed to Schistosoma mansoni cercariae and the infection monitored until portal perfusion at 18 weeks. Despite a wide variation in worm numbers recovered, fecal egg output and circulating antigen levels indicated that a substantial population had established in all animals. Half the macaques had portal hypertension but only one had portacaval shunts, ruling out translocation to the lungs as the reason for loss of adult burden. Many worms had a shrunken and pallid appearance, with degenerative changes in intestines and reproductive organs. Tegument, gut epithelia and muscles appeared cytologically intact but the parenchyma was virtually devoid of content. An early and intense IgG production correlated with low worm burden at perfusion, and blood-feeding worms cultured in the presence of serum from these animals had stunted growth. Using immunoproteomics, gut digestive enzymes, tegument surface hydrolases and antioxidant enzymes were identified as targets of IgG in the high responder animals.nnnSIGNIFICANCEnIt appears that worms starve to death after cessation of blood feeding, as a result of antibody-mediated processes. We suggest that proteins in the three categories above, formulated to trigger the appropriate mechanisms operating in rhesus macaques, would have both prophylactic and therapeutic potential as a human vaccine.

Antimicrobial functions of mononuclear phagocytes

One of the major functions of mononuclear phagocytes, i.e., monocytes and macrophages, is the phagocytosis and killing of microorganisms. To obtain more insight into the pathogenesis of infectious diseases and to develop new therapies against these diseases, a better understanding of the antimicrobial mechanisms employed by mononuclear phagocytes is essential. The present review gives a short description of the mononuclear phagocyte system and summarizes various methods that are used to study the antimicrobial mechanisms of mononuclear phagocytes.

Nitrite Production by Activated Murine Macrophages Correlates with Their Toxoplasmastatic Activity, Ia Antigen Expression, and Production of H2O2

Activated macrophages have various characteristics in common with exudate and resident macrophages, but the ability to inhibit intracellular proliferation of the protozoa Toxoplasma gondii, the expression of Ia antigen and the capacity to produce H2O2 varies among these cells. Assessment of these features of macrophages, which are generally used as criteria for macrophage activation, has certain drawbacks. Since activated murine macrophages, but not exudate or resident macrophages, produce considerable amounts of NO2-, assessment of NO2- production by these cells might serve as a measure of macrophage activation. The aim of the present study was to find out whether NO2- production by murine peritoneal macrophages correlates with the three generally accepted criteria for macrophage activation. Quantitative data on resident, exudate and activated macrophages revealed that the production of NO2- stimulated by a calcium-ionophore correlates best with the ability to inhibit the proliferation of T. gondii, Ia antigen expression, and capacity to produce H2O2. Because it is rapid and easy to perform, measurement of the amount of NO2- produced by murine macrophages stimulated with a calcium-ionophore offers the most practical criterion for distinction between activated macrophages and exudate and resident macrophages.

Flow cytometric analysis on reactivity of human T lymphocyte-specific and cytokine-receptor-specific antibodies with peripheral blood mononuclear cells of chimpanzee (Pan troglodytes), rhesus macaque (Macaca mulatta), and squirrel monkey (Saimiri sciureus)

Abstract: There are relatively few monoclonal antibodies (mAb) that have been characterized for their applicability in studies on the immune system of various nonhuman primates. In the present study, we identified a large number of mAb that can be used in future immunological studies in three different nonhuman primates, i.e., chimpanzees, rhesus macaques, and squirrel monkeys. The reactivity of 161 anti‐human mAb to T‐cell antigens and cytokine receptors were tested on peripheral blood mononuclear cells (PBMC) from the three primate species by flow cytometric analysis. A total of 105 (65%), 73 (45%), and 68 (42%) antibodies reacted with PBMC from chimpanzees, rhesus macaques, and squirrel monkeys, respectively. Out of the 161 mAb, 38 reacted with all three species and 112 reacted with one or two of the species. No specific reaction was observed with mAb to receptors to GM‐CSF, 4–1BB, FLT3, FLX2, common β‐chain, IL‐1 (type I receptor), and IL‐8.

Safety evaluation of a polymerized hemoglobin solution in a murine infection model

Several investigators have observed that free hemoglobin may increase the mortality rate in experimental Escherichia coli peritonitis in animals. This effect is probably mediated by the heme moiety of hemoglobin, but the mechanism remains controversial. Free hemoglobin might impair neutrophil function, and it might serve as a source of iron, which is necessary for bacterial replication. Several modified hemoglobin solutions, developed as blood substitutes, are currently being tested in clinical studies, but concern exists that these solutions may have the potential to exacerbate a bacterial infection. At the Central Laboratory of the Netherlands Red Cross Blood Transfusion Service, a blood substitute based on modified hemoglobin (PolyHbXl) has been developed that has improved oxygen affinity and prolonged vascular retention. In the present study the potential risk of this solution on the promotion of infections has been evaluated. PolyHbXl was intravenously injected into mice in a clinically relevant dose of 1.5 gm/kg body weight 1 hour before intravenous administration of a sublethal number of Listeria monocytogenes, Salmonella typhimurium, E. coli, or Candida albicans organisms. PolyHbXl did not promote the proliferation of any of these microorganisms in the liver and spleen, nor did it lead to an increased mortality rate in the mice. Also, the in vitro proliferation of L. monocytogenes, S. typhimurium, and E. coli was not increased by PolyHbXl. In conclusion, PolyHbXl does not affect the course of infection with various microorganisms in mice, and no indication was found that this new blood substitute compromises the host defense against infections.