The IsTaR 1 serodeme of Trypanosoma brucei: development of a new serodeme

An extensive serodeme of sequentially-isolated antigenic variants of African trypanosomes has been produced from both syringe-passaged and cyclically-transmitted Trypanosoma brucei of the IsTaR 1 clone derived from EATRO 164. The majority of the antigenic variants were isolated from chronically-infected deer mice (Peromyscus leucopus). The pattern of parasitemias during the course of infections initiated with syringe-passaged trypanosomes differed from those initiated with cyclically-transmitted trypanosomes. Trypanosome populations from syringe-passaged (192) and cyclically-transmitted (31) clones were each amplified by growth in lethally-irradiated mice and cryopreserved for retrospective analysis. Five clones derived from a single deer mouse during the first 44 days of infection, and 2 clones derived from an acutely-infected rat were established from these amplified populations. Homogeneous populations were grown in lethally-irradiated rats and mice for antigenic analysis purification of variant-specific glycoprotein. Six of the 7 clones were distinct variants by immunological criteria using antisera derived from whole cells or purified surface glycoproteins. Two clones, one derived from the acutely-infected rat, and the other from the first parasitemia in a chronic infection that was initiated with the former clone, were immunologically identical. Production of these clones established a well-defined serodeme that will allow detailed analysis of antigenic variation.     

Immune responses during human schistosomiasis mansoni. VI. In vitro nonspecific suppression of phytohemagglutinin responsiveness induced by exposure to certain schistosomal preparations.

Simultaneous in vitro exposure of human peripheral blood mononuclear cells to phytohemagglutinin-P (PHA) and either soluble schistosomal egg antigenic preparation (SEA) or soluble cercarial antigenic preparation (CAP) obtained from Schistosoma mansoni resulted in decreased responsiveness as compared to exposure to PHA alone. The addition of a soluble adult worm antigenic preparation (SWAP) did not predictably alter PHA responses in this system. The suppression due to in vitro exposure to either SEA or CAP was expressed whether the lymphocyte donors were S. mansoni patients (early infection, chronic, or treated), or uninfected subjects. The degree of suppression was related to the concentration of SEA used, and the timing of exposure. Preexposure to SEA for 3 days before the addition of PHA resulted in more potent suppression. However, a delay in the time of the addition of SEA of 6 and 24 hr after PHA exposure decreased and eliminated, respectively, its suppressive capacity. SEA and CAP were not directly toxic to responding cells, and appeared to exert their nonspecific suppressive influences through T lymphocyte-related mechanisms. It was observed that although these suppressive events could be induced and observed in vitro, the responsiveness of S. mansoni patient lymphocytes to PHA was equal with that of uninfected controls.     

Conserved Immune Recognition Hierarchy of Mycobacterial PE/PPE Proteins during Infection in Natural Hosts

The Mycobacterium tuberculosis genome contains two large gene families encoding proteins of unknown function, characterized by conserved N-terminal proline and glutamate (PE and PPE) motifs. The presence of a large number of PE/PPE proteins with repetitive domains and evidence of strain variation has given rise to the suggestion that these proteins may play a role in immune evasion via antigenic variation, while emerging data suggests that some family members may play important roles in mycobacterial pathogenesis. In this study, we examined cellular immune responses to a panel of 36 PE/PPE proteins during human and bovine infection. We observed a distinct hierarchy of immune recognition, reflected both in the repertoire of PE/PPE peptide recognition in individual cows and humans and in the magnitude of IFN-γ responses elicited by stimulation of sensitized host cells. The pattern of immunodominance was strikingly similar between cattle that had been experimentally infected with Mycobacterium bovis and humans naturally infected with clinical isolates of M. tuberculosis. The same pattern was maintained as disease progressed throughout a four-month course of infection in cattle, and between humans with latent as well as active tuberculosis. Detailed analysis of PE/PPE responses at the peptide level suggests that antigenic cross-reactivity amongst related family members is a major determinant in the observed differences in immune hierarchy. Taken together, these results demonstrate that a subset of PE/PPE proteins are major targets of the cellular immune response to tuberculosis, and are recognized at multiple stages of infection and in different disease states. Thus this work identifies a number of novel antigens that could find application in vaccine development, and provides new insights into PE/PPE biology.     

Some aspects of the immunology of pseudotuberculosis

Integral evaluation of some humoral and cellular immunity factors in patients with pseudotuberculosis (Far-East scarlatinoid fever) with varying severity of its course was carried out. Inadequate immune reaction of human organism in response to infection with pseudotuberculosis was established at the level of both specific (sporadic cases of lacking synthesis or poor synthesis of IgM antibodies, lacking synthesis of IgG antibodies) and non-specific factors (incomplete phagocytic reaction). Phagocytosis is higher in monocytes than in neutrophils during pseudotuberculosis. The opsonizing properties of blood serum, the intensity of which increases in the course of disease, are great importance in freeing the organism from bacteria of pseudotuberculosis. The most rapid and technically simplest is the determination of antibodies in blood serum of the patients by means of the reaction of indirect hemagglutination. For this purpose the authors developed a new, highly sensitive, and specific preparation - the dry erythrocyte antigenic diagnostic agent.     

Rapid Detection of Contaminated Intravenous Fluids Using the Limulus In Vitro Endotoxin Assay

Intravenous fluids and administration sets may become contaminated with gram-negative bacteria during use and result in a life-threatening situation to the patient. The Limulus in vitro assay for endotoxin was used in two patients whose parenteral fluids had become contaminated with Pseudomonas aeruginosa. This test allowed rapid detection of the contaminated intravenous fluids and demonstrated a concomitant endotoxemia in both patients. The same strains of pseudomon were subsequently cultured from each patient's blood, intravenous catheter tip, and parenteral fluid and administration set. A different serotype of pseudomonas was unique to each patient, indicating two separate and unrelated cases of accidental contamination of the administration sets. Endotoxin-like activity was also demonstrated from several brands of commercial human serum albumin, which may contribute low-level activity detectable by the Limulus assay.     

Establishment and characterization of NS3 protein-specific T-cell clones from a patient with chronic hepatitis C

Our previous study showed dominant proliferative response of peripheral mononuclear cells to hepatitis C virus (HCV) nonstructural (NS-3) (T9, from aa 1188 to 1493) in chronically infected patients. Six T9-specific T-cell clones derived in an HCV patient were established and studied for the antigen specificity and the ability of augmentation of in vitro antibody production. All these cloned T-cell lines responded exclusively to T9 antigen and could help autologous B cells in producing anti-T9 antibody in vitro. Cytokine mRNAs of these T cells was detected by polymerase chain reaction and predominant IL-2 and IFN- production was noted. In addition, further elucidation of T-cell antigenic determinant and MHC restriction suggested that these T-cell clones recognized at least two different T-cell antigenic determinants within the NS-3 region in an HLA DQ2-restricted manner. We believe characterization of HCV-specific T-cell responses, especially T-cell epitope mapping and cytokine production pattern, may shed light on further understanding the pathogenic mechanism and designing therapy for HCV infection.     

Selection of a Borrelia burgdorferi antigenic variant by cultivation in the presence of increasing amounts of homologous immune serum

This investigation was undertaken to select antigenic variants of a Borrelia burgdorferi strain in vitro. The original strain BITS was cultivated in BSK medium supplemented with increasing concentrations of homologous hyperimmune serum raised in rabbits. After a few serial passages starting from a subinhibitory serum dilution of 1:800 in BSK up to 1:200, a variant named BITSv was obtained; it grew abundantly like the control culture in the presence of hyperimmune serum. Analysis of the antigenic pattern of the original and derived variants by Western blotting revealed that BITSv, compared to the original strain BITS, had lost the reactivity with the immune serum at the level of the oligosaccharide moiety. These experiments, designed to mimic the possible action of antibodies that arise during a Borrelia infection, suggest that lipopolysaccharides are surface located and that they play a role in the integrity of the outer membrane during the multiplication of Borrelia burgdorferi.     

Identification of antigenic regions of duck hepatitis B virus core protein with antibodies elicited by DNA immunization and chronic infection

The induction of humoral response in ducks by DNA-based immunization against duck hepatitis B virus (DHBV) core protein (DHBc) was investigated. In addition, the amino acid specificity of the induced response was compared by using peptide scanning to that elicited either by protein immunization or during chronic DHBV infection. Immunization of ducks with a plasmid expressing DHBc protein led to the induction of a long-lasting antibody response able to specifically recognize viral protein in chronically infected duck livers. Peptide scanning analysis of anti-DHBc response induced during chronic DHBV infection allowed us to identify six major antigenic regions (AR1 to AR6). The reactivity spectrum of duck sera elicited by protein immunization appeared narrower and was restricted to only four of these antigenic regions in spite of higher anti-DHBc antibody titers. Interestingly, anti-DHBc antibodies induced by DNA-based immunization recognized five of six antigenic regions, and the epitope pattern was broader and more closely related to that observed in chronic viral infections. To gain more insight into the location of antigenic regions, we built a three-dimensional (3-D) model of DHBc protein based on human and duck core sequence alignment data and the HBc 3-D crystal structure. The results suggest that two identified antigenic regions (AR2, amino acids [aa] (64)T-P(84), and AR5, aa (183)A-R(210)) are located at positions on the protein surface equivalent to those of the two HBc major epitopes. Moreover, we identified another antigenic region (AR3, aa (99)I-I(112)) that was recognized by all sera from chronically infected, DNA- or protein-immunized ducks within the large 45-aa insertion in DHBc protein, suggesting that this region, which lacks HBc, is externally exposed.     

Variability of HIV infections.

Genetic variation is the hallmark of infections with lentiviruses in general and the human immunodeficiency viruses (HIV-1, HIV-2) in particular. This article reviews both experimental evidence for the variability of the HIV genome during the course of an individual infection and mathematical models that outline the potential importance of antigenic variation as a major factor to drive disease progression. The essential idea is that the virus evades immune pressure by the continuous production of new mutants resistant to current immunological attack. This results in the accumulation of antigenic diversity during the asymptomatic period. The existence of an antigenic diversity threshold is derived from the asymmetric interaction between the virus quasispecies and the CD4 cell population: CD4 cells mount immune responses some of which are directed against specific HIV variants, but each virus strain can induce depletion of all CD4 cells and therefore impair immune responses regardless of their specificity. Therefore, increasing HIV diversity enables the virus population to escape from control by the immune system. In this context the observed genetic variability is responsible for the fact that the virus establishes a persistent infection without being cleared by the immune response and induces immunodeficiency disease after a long and variable incubation period. Mathematical biology has revealed a novel mechanism for viral pathogenesis.     

The B cell response is redundant and highly focused on V1V2 during early subtype C infection in a Zambian seroconverter

High-titer autologous neutralizing antibody responses have been demonstrated during early subtype C human immunodeficiency virus type 1 (HIV-1) infection. However, characterization of this response against autologous virus at the monoclonal antibody (MAb) level has only recently begun to be elucidated. Here we describe five monoclonal antibodies derived from a subtype C-infected seroconverter and their neutralizing activities against pseudoviruses that carry envelope glycoproteins from 48 days (0 month), 2 months, and 8 months after the estimated time of infection. Sequence analysis indicated that the MAbs arose from three distinct B cell clones, and their pattern of neutralization compared to that in patient plasma suggested that they circulated between 2 and 8 months after infection. Neutralization by MAbs representative of each B cell clone was mapped to two residues: position 134 in V1 and position 189 in V2. Mutational analysis revealed cooperative effects between glycans and residues at these two positions, arguing that they contribute to a single epitope. Analysis of the cognate gp120 sequence through homology modeling places this potential epitope near the interface between the V1 and V2 loops. Additionally, the escape mutation R189S in V2, which conferred resistance against all three MAbs, had no detrimental effect on virus replication in vitro. Taken together, our data demonstrate that independent B cells repeatedly targeted a single structure in V1V2 during early infection. Despite this assault, a single amino acid change was sufficient to confer complete escape with minimal impact on replication fitness.     

Experimental investigation of changes in the antigenic structure of influenza viruses during their persistence

Systems of persistent influenza infection produced in the cultures of human embryonal kidneys and in the diploid cells of human embryonal lungs under the effect of A/Hong-Kong/1/68 and A/Victoria/35/72 viruses were characterized. The cell systems were studied in the course of 4--3u passages, i. e., from 40 to 289 days. A total of 102 viruses were isolated during the period of observation, out of which 44 were examined for the antigenic profile of HA and NA. The antigenic structure of the initial viruses was preserved in 31 variations isolated while in 13 variants it was pronouncedly and permanently changed. The following mechanisms of the mentioned phenomenon are discussed: mutations, intrapopulation recombinations and integrations.     

Epidemiological studies on Japanese encephalitis in Kyoto City area, Japan. III. Seasonal prevalence of virus infections in several pig populations shown by virus recovery from engorged Culex tritaeniorhynchus summorosus.

When vector mosquitoes engorged by feeding on pigs are tested for virus recovery after incubation for 7 to 10 days, the results may show mosquito infection itself. Therefore, seasonal prevalence of infection in each pig population can be estimated from course of the infection rate among mosquitoes. Many mosquitoes of the main vector of Japanese encephalitis virus, Culex tritaeniorhynchus summorosus, collected by light traps everyday or every other day in some pig sheds from 1967 to 1970 were tested for virus recovery after incubation. The tests were positive during about a month period each summer, and the peak infection rate was high being over 10%. The course of the mosquito infection showed a certain pattern with one or two peaks between the initial recovery and the highest peak. From the interval of 12 to 13 days after the first recovery to the peak, cyclic infection between the pig and the mosquito may occur at the same interval.     

Chagas disease and HIV co-infection: genetic analyses of two <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis>strains under experimental immunosuppression

BACKGROUND: Recently new aspects of the immunopathology of Chagas disease have been described in patients infected with HIV and unusual clinical manifestations such as cutaneous lesions, involvement of central nervous system and/or serious cardiac lesions related to the reactivation of the parasite have been reported. Two uncloned Trypanosoma cruzi strains previously isolated from chronic chagasic patients with HIV co-infection were studied in order to evaluate the impact of the immunosuppression on the genetic diversity of the parasite. RESULTS: We have exploited an experimental model to determine whether genetically distinct populations appear after immunosuppression as a consequence of in vivo selection or in vitro propagation. The in vitro and in vivo conditions have allowed us to study the selected populations. The first strain was isolated from a case of reactivation of Chagas disease in a patient which presented four cerebral lesions. It was possible to demonstrate that the patient was infected with at least three distinct populations of T. cruzi. The population, recovered after immunosuppression, in mice was genetically divergent from the primary human isolate. The second strain, isolated from a hemophiliac/HIV positive patient presenting cardiac manifestation of Chagas disease showed no marked genetic difference after experimental immunosuppression. CONCLUSION: The immunological condition of the patient, associated or not to the reactivation of the infection, and also the strain of the parasite may have an important role during the course of the disease. The in vivo mechanism that generates parasite genetic variability or the participation of the selection under stress conditions will require further investigation.     

Antigenic and genetic variation in cytopathic hepatitis A virus variants arising during persistent infection: evidence for genetic recombination.

Variants of hepatitis A virus (pHM175 virus) recovered from persistently infected green monkey kidney (BS-C-1) cells induced a cytopathic effect during serial passage in BS-C-1 or fetal rhesus kidney (FRhK-4) cells. Epitope-specific radioimmunofocus assays showed that this virus comprised two virion populations, one with altered antigenicity including neutralization resistance to monoclonal antibody K24F2, and the other with normal antigenic characteristics. Replication of the antigenic variant was favored over that of virus with the normal antigenic phenotype during persistent infection, while virus with the normal antigenic phenotype was selected during serial passage. Viruses of each type were clonally isolated; both were cytopathic in cell cultures and displayed a rapid replication phenotype when compared with the noncytopathic passage 16 (p16) HM175 virus which was used to establish the original persistent infection. The two cytopathic virus clones contained 31 and 34 nucleotide changes from the sequence of p16 HM175. Both shared a common 5' sequence (bases 30 to 1677), as well as sequence identity in the P2-P3 region (bases 3249 to 5303 and 6462 to 6781) and 3' terminus (bases 7272 to 7478). VP3, VP1, and 3Cpro contained different mutations in the two virus clones, with amino acid substitutions at residues 70 of VP3 and 197 and 276 of VP1 of the antigenic variant. These capsid mutations did not affect virion thermal stability. A comparison of the nearly complete genomic sequences of three clonally isolated cytopathic variants was suggestive of genetic recombination between these viruses during persistent infection and indicated that mutations in both 5' and 3' nontranslated regions and in the nonstructural proteins 2A, 2B, 2C, 3A, and 3Dpol may be related to the cytopathic phenotype.     

Cell-mediated immune response in human hepatic echinococcosis. Analysis of antigen-induced lymphoproliferation and NK activity

In vitro cellular immune responsiveness was studied in 25 patients undergoing surgery for hepatic hydatid disease and in 22 matched healthy controls. Proliferation of peripheral blood mononuclear cells (PBMC) induced by phytohaemagglutinin (PHA) was not statistically different in the two groups, while proliferation induced by antigenic preparations obtained from the human commensal microorganism Candida albicans was depressed in patients as compared to healthy subjects. Confirming previous data, antigen specific proliferative response to hydatid cyst fluids was greatly enhanced in patients as compared to controls (P less than 0.01). On the other hand, natural killer (NK) activity was significantly reduced (P less than 0.005). Both impairment of NK activity and lymphoproliferation induced by commensal microorganisms suggest that patients following the parasitic infection present a condition of relevant hyporesponsiveness in cell-mediated defence.     

Antigenic diversity among Portuguese clinical isolates of Helicobacter pylori

Background: The human gastroduodenal pathogen, Helicobacter pylori, is characterized by an unusual extent of genetic heterogeneity. This dictates differences in the antigenic pattern of strains resulting in heterogeneous human humoral immune responses. Here, we examined the antigenic variability among a group of 10 strains isolated from Portuguese patients differing in age, gender, and H. pylori‐associated gastric diseases. Material and Methods: Immunoassays were performed on two‐dimensional electrophoresis gels obtained for the proteome of each strain, using a commercial pool of antibodies produced in rabbit, against the whole cell lysate of an Australian H. pylori strain. Relevant proteins were identified by mass spectrometry. Results: Immunoproteomes of the Portuguese strains showed no correlation between the number of antigenic proteins or the antigenic profile, and the disease to which each strain was associated. The Heat shock protein B was the unique immunoreactive protein common to all of them. Additionally, seven proteins were found to be antigenic in at least 80% of strains: enoyl‐(acyl‐carrier‐protein) reductase (NADH); Catalase; Flagellin A; 2 isoforms of alkyl hydroperoxide reductase; succinyl‐CoA transferase subunit B; and an unidentified protein. These proteins were present in the proteome of all tested strains, suggesting that differences in their antigenicity are related to antigenic variance. Conclusions: This study showed evidence of the variability of antigenic pattern among H. pylori strains. We believe that this fact contributes to the failure of anti‐H. pylori vaccines and the low accuracy of serological tests based on a low number of proteins or antigens of only one strain.     

Secreted antigens of the amastigote and promastigote forms of Leishmania infantum inducing a humoral response in humans and dogs.

To study the antigens secreted by promastigote and amastigote forms of Leishmania infantum which are able to induce a humoral response in human patients and dogs, we have carried out immunoprecipitation assays with different supernatants of in vitro cultured parasites, metabolically labelled with [35S]methionine, using serum samples from human patients and dogs. In addition, some metabolic labelling experiments were performed daily during the in vitro culture parasite's life cycle to follow the time course excretion-secretion of parasitic antigens. The results demonstrated that the two different hosts developed an antibody response against secreted antigens of both stages of Leishmania infantum. Nevertheless, the humoral response directed against the excreted-secreted antigens of the promastigote forms was qualitatively and quantitatively different when we compare the human and the dog immune responses. On the other hand, when the excreted-secreted antigens of the amastigote forms are immunoprecipitated with either human or canine immune serum, the humoral response is similar. In addition, the time course study showed that excretion-secretion of antigens was qualitatively and quantitatively modulated during the parasitic in vitro life cycle.     

Plasmodium falciparum infection causes proinflammatory priming of human TLR responses

TLRs are a major group of pattern recognition receptors that are crucial in initiating innate immune responses and are capable of recognizing Plasmodium ligands. We have investigated TLR responses during acute experimental P. falciparum (P.f.) infection in 15 malaria-naive volunteers. TLR-4 responses in whole blood ex vivo stimulations were characterized by significantly (p < 0.01) up-regulated proinflammatory cytokine production during infection compared with baseline, whereas TLR-2/TLR-1 responses demonstrated increases in both proinflammatory and anti-inflammatory cytokine production. Responses through other TLRs were less obviously modified by malaria infection. The degree to which proinflammatory TLR responses were boosted early in infection was partially prognostic of clinical inflammatory parameters during the subsequent clinical course. Although simultaneous costimulation of human PBMC with P.f. lysate and specific TLR stimuli in vitro did not induce synergistic effects on cytokine synthesis, PBMC started to respond to subsequent TLR-4 and TLR-2 stimulation with significantly (p < 0.05) increased TNF-alpha and reduced IL-10 production following increasing periods of preincubation with P.f. Ag. In contrast, preincubation with preparations derived from other parasitic, bacterial, and fungal pathogens strongly suppressed subsequent TLR responses. Taken together, P.f. primes human TLR responses toward a more proinflammatory cytokine profile both in vitro and in vivo, a characteristic exceptional among microorganisms.     

In vivo expression of the heat stable (estA) and heat labile (eltB) toxin genes of enterotoxigenic Escherichia coli (ETEC)

Enterotoxigenic Escherichia coli (ETEC) colonize the intestine and adhere to the epithelium by means of different host specific colonization factors (CFs). Colonizing ETEC produce one or both of two enterotoxins; the heat stable (ST) and heat labile (LT) toxins which are both able to cause diarrhoea. The regulation of virulence genes in ETEC during infection of the human intestine is mainly unknown. In this study we analysed the level of mRNA expression of estA, coding for ST, and eltB, coding for the B subunit of LT, during human infection. The expressions of the toxins in ETEC strains expressing both ST and LT were investigated in bacteria isolated directly from patient stool without sub-culturing, (in vivo) and compared to the expression pattern of the corresponding ST/LT strains grown in liquid broth (in vitro) by quantitative competitive RT-PCR using fluorescent primers. We found that estA and eltB are expressed in the in vivo samples but no significant up-or down regulation of the expression levels of either estA or eltB could be determined in vivo as compared to in vitro.