Longitudinal serum HIV RNA quantification: correlation to viral phenotype at seroconversion and clinical outcome.

OBJECTIVE: To investigate the longitudinal changes in serum HIV RNA, and to clarify whether the viral load early in infection has a predictive value for the clinical outcome; also, to correlate viral phenotype at seroconversion and changes in CD4 cell counts with viral burden. DESIGN: Twenty seroconverters with HIV isolates available at seroconversion had HIV RNA quantified by polymerase chain reaction (PCR) at seroconversion and thereafter every 6 months. Mean follow-up time was 65 months. Patients were classified according to viral phenotype at seroconversion, time to AIDS progression, serum viral load within the first year (less or more than 1.5 x 10(4) copies/ml). RESULTS: High viral load at seroconversion was followed by a significant decline within the first months (P < 0.0005). Decline to < 1.5 x 10(4) copies/ml was correlated with slower progression to AIDS (P < 0.05). A correlation between the rate of CD4 decline and the median viral load during the ensuing viral load plateau phase was also shown (P < 0.05). Subsequent to this phase the viral burden increased. Rapid progressors had higher viral load than slow- or non-progressors; this was particularly pronounced late in infection. Harbouring syncytium-inducing (SI) virus at seroconversion was associated with faster progression to AIDS than non-SI (NSI; P < 0.005). The increased in vitro replication rate of SI over NSI was not translated into significantly higher serum HIV RNA. CONCLUSION: Serum HIV RNA is high around the time of seroconversion. A significant decline within the first months hereafter is followed by a plateau phase, which in turn is followed by an increase in HIV RNA. HIV RNA early in infection has a predictive value for the clinical outcome. The increased virulence of SI over NSI virus did not translate into significantly higher HIV RNA values.

Antibody reactivity to conserved linear epitopes of Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1).

The Plasmodium falciparum erythrocyte membrane protein 1 (PfEMP1) family of protein antigens are involved in adhesion of P. falciparum infected erythrocytes to the capillary endothelium of the host. Antibodies to variable regions of these proteins, measured by agglutination, correlates with clinical protection against falciparum malaria. In this study we investigated the occurrence of antibodies to conserved sequences of these very variable proteins in a population living in an area endemic for falciparum malaria. Using the ELISA method, we were able to measure an antibody response to three synthetic peptides derived from conserved regions of PfEMP1. The antibody responses to these peptides increased with age and were higher in individuals with asymptomatic P. falciparum infection compared to individuals presenting with fever attributable to falciparum malaria. This indicates that antibodies recognising the conserved regions of PfEMP1 arise upon exposure to the parasite, and that these may be involved in the development of protection against malaria. Antibodies to the Pfalhesin peptide of the human aniontransporter, band3, were measured by the same method. The magnitude of this antibody response did not correlate with neither age nor clinical protection.

Soluble haemoglobin is a marker of recent Plasmodium falciparum infections.

Monoclonal antibodies (Mab) were raised against haemoglobin (Hb) associated with Plasmodium falciparum protein and used to develop an ELISA, measuring circulating levels of released Hb. This assay was evaluated in different malaria patients in parallel with ELISA assays for C-reactive protein (CRP) and haptoglobin. Levels of Hb were negatively associated with levels of haptoglobin. Increased levels of serum Hb and CRP and decreased levels of haptoglobin were seen in Danish malaria patients. Consecutive studies showed that increased Hb levels were detectable 3-7 days after initiation of treatment probably because of drug induced destruction of infected erythrocytes. Increased levels of CRP were measured 0-3 days after initiation of treatment. The Hb assay was used in an epidemiological study of malaria in an area of Sudan with unstable malaria transmission. The proportion of Sudanese adults with detectable soluble Hb was higher in the rainy season with malaria transmission compared to the dry season. Hb levels in the rainy season were negatively associated with levels of haptoglobin. Most adults had increased levels of soluble Hb and decreased levels of haptoglobin 7 and 30 days after their treatment of P. falciparum malaria compared to the levels during acute disease. Thus, both soluble Hb and haptoglobin appear to be markers of recent P. falciparum infections. Very high levels of CRP protein were measured in some of the malaria patients at the day of treatment while lower levels were recorded 7 and 30 days after treatment. Soluble Hb levels were associated with malariometric parameters in a similar fashion to haptoglobin. The new Mab-based assay for measuring soluble Hb in the peripheral blood of malaria patients may be useful for future epidemiological studies of malaria.

Reduced cellular immune reactivity in healthy individuals during the malaria transmission season.

Antigen-induced cellular immune responses are suppressed during acute malaria. The present study engages the possibility that malaria-induced alterations in cellular immune reactivity extend beyond the clinical disease. Thus, lymphoproliferative responses of healthy individuals were diminished during the malaria transmission period in individuals living in an area of highly seasonal, unstable malaria transmission. This finding may have important implications for the design of studies of stimulatory properties of antigens using lymphocytes of endemic origin.

Serine-stretch protein (SERP) of Plasmodium falciparum corresponds to the exoantigen Ag2, a target of antibodies associated with protection against malaria.

A mixture of Plasmodium falciparum exoantigens inducing lymphocyte activation and cytokine production was shown to contain the malaria vaccine candidate, the serine-stretch protein. This protein was shown serologically to correspond to Ag2, an exoantigen recognized by antibodies linked with protection against malaria. The glycophorin-binding protein, the histidine-rich protein II, the S-antigen, the heat shock protein 70, the ring-infected erythrocyte surface antigen, and the apical membrane antigen-1 were also shown serologically to be present in the mixture of exoantigens.

Decreased plasma levels of factor II + VII + X correlate with increased levels of soluble cytokine receptors in patients with malaria and meningococcal infections.

The levels of coagulation factors II + VII + X and of blood platelets (thrombocytes) as well as of cytokines and soluble cytokine receptors were studied in the patients with malaria or meningococcal infections. The coagulation factors were decreased particularly in the meningococcal patients, while thrombocytes were lowest in the Plasmodium falciparum malaria patients. There was no correlation between factors II + VII + X and thrombocytes, but plasma levels of coagulation factors II + VII + X were found to correlate inversely with levels of soluble interleukin-2 receptor (sIL-2R) and soluble tumour necrosis factor-I (sTNF-RI) in patients with malaria and meningococcal infections. Elevated sIL-2R and sTNF-RI levels and decreased coagulation factors reverted to normal within 3-5 days after initiation of therapy in P. falciparum patients followed consecutively. Estimation of coagulation factors may be used to monitor the course of these common and potentially life-threatening infections.

T-cell responses in malaria.

Malaria is caused by infection with protozoan parasites of the genus Plasmodium. It remains one of the most severe health problems in tropical regions of the world, and the rapid spread of resistance to drugs and insecticides has stimulated intensive research aimed at the development of a malaria vaccine. Despite this, no efficient operative vaccine is currently available. A large amount of information on T-cell responses to malaria antigens has been accumulated, concerning antigens derived from all stages of the parasite life cycle. The present review summarizes some of that information, and discusses factors affecting the responses of T cells to malaria antigens.

Isolation and characterization of a soluble antigen complex of Plasmodium falciparum with pyrogenic properties.

A soluble antigen complex, previously designated antigen no. 7 (Ag7) on the basis of the pattern obtained by crossed immunoelectrophoresis of culture supernatants of P. falciparum, was isolated by affinity chromatography. It was shown to be synthesized at the schizont stage of the parasite growth cycle and to be located on the surface of the schizonts. Antibodies to Ag7 did not inhibit the growth of the parasite in vitro. Ag7 is recognized by immune human sera from many parts of the world and it stimulated the production of specific antibody in mice when incorporated into immune-stimulating complex (ISCOM) structures. It also specifically stimulated in vitro proliferation of lymphocytes from clinically immune adults. That it induced the secretion of interleukin 1 by human monocytes and was pyrogenic in rabbits was of particular interest. Thus Ag7 has endotoxin-like properties which make it a possible candidate for an antitoxic malaria vaccine.

Identification and localization of a soluble antigen, Ag2, of 136 kDa from Plasmodium falciparum in vitro cultures.

The soluble antigens, antigen 2 (Ag2) and antigen 6 (Ag6), were copurified from supernatants of P. falciparum in vitro cultures by affinity chromatography and Fast Protein Liquid Chromatography. Rabbit antibodies to Ag2 were raised and characterized by crossed immunoelectrophoresis. Ag2 appeared as a duplet with molecular masses of 136 and 120 kDa when tested by immunoblotting. Immunoprecipitation experiments on Triton X-100 extracted antigens from synchronized cultures showed that the antigen was synthesized in the schizont stage. Ag2 was located near the surface of schizonts in the parasitophorous vacuole and in clefts in the infected erythrocyte cytoplasma as shown by immunogold electron microscopy.

Cell-mediated immune responses to soluble Plasmodium falciparum antigens in residents from an area of unstable malaria transmission in the Sudan.

This paper describes immune responses to P. falciparum infection in individuals living in an area of highly seasonal, unstable malaria transmission. The in vitro cellular immune responses of peripheral blood mononuclear cells (PBMC) from 36 Sudanese donors to a complex of affinity purified soluble P. falciparum antigens (SPag) and two components thereof (Ag1 and Ag7) were examined and compared to humoral immune parameters. In 29/36 Sudanese donors, SPag induced a significant lymphoproliferative response in vitro. In contrast only 3/27 Danish donors never exposed to malaria responded to SPag. Ag1 and Ag7 induced significant lymphoproliferation in 9/34 and 13/36 Sudanese donors respectively, whereas no Danish donors responded. Significant interferon-gamma production was observed in 16/27, 1/5 and 3/12 Sudanese donors when stimulated by SPag, Ag1 and Ag7 respectively. Lymphoproliferative responses to SPag correlated with proliferative responses to Ag1 and Ag7, and with Spag-induced interferon-gamma production. These results indicate that T-cell clones recognizing epitopes on Ag1 and Ag7 have been expanded in the studied population as a result of exposure to P. falciparum infection. The T-cell parameters did not correlate with the presence of antibodies to SPag, Pf155/RESA or a crude parasite sonicate or with the schizont IFA titers of the plasma. This indicates that parameters outside the degree of exposure to P. falciparum influence the cellular immune responses to malaria.

Antibody reactivities to glutamate-rich peptides of Plasmodium falciparum parasites in humans from areas of different malaria endemicity.

Synthetic P. falciparum peptides were evaluated as tools in epidemiological investigations of malaria. Plasma IgM and IgG antibody reactivities against synthetic peptides covering sequences of glutamate-rich protein (GLURP) and acidic-basic repeat antigen (ABRA) were measured by ELISA in individuals from malaria-endemic areas of Sudan, Indonesia and The Gambia to study antibody responses to these peptides in donors living in areas of different malaria endemicity. IgG and IgM reactivities to the peptides increased with malaria endemicity, although there were no differences in reactivities to the GLURP peptide between non-exposed donors and donors living in areas of low malaria endemicity. IgG reactivities to the GLURP peptide in Sudanese adults were high one month after treatment in all adults tested, while IgG reactivities to the ABRA peptide were infrequent. IgM responses to the peptides tested were shortlived in most patients. In Gambian children with malaria, IgM reactivities but not IgG antibody reactivities against the ABRA peptide were higher in those with mild malaria than in those with severe malaria. The peptides may be useful in future epidemiological studies, especially in areas of low malaria endemicity.

Bancroftian filariasis: the patterns of filarial-specific immunoglobulin G1 (IgG1), IgG4, and circulating antigens in an endemic community of northeastern Tanzania.

The profile of filarial-specific immunoglobulin G1 (IgG1), IgG4, and Wuchereria bancrofti-specific circulating antigen (Og4C3) was analyzed in individuals one year of age and older in a community with high endemicity for Bancroftian filariasis. The overall microfilarial (mf) prevalence in the examined population was 29%. Fifty-one percent of the population were positive for IgG1 (39% among mf-positive individuals and 63% among mf-negative individuals), whereas 90% were positive for IgG4 (97% among mf-positives and 87% among mf-negatives). The levels of IgG1 and IgG4 were clearly related to mf status and age, but they were unrelated to sex, intensity of microfilaremia, or chronic clinical manifestations. The mean level of IgG1 was significantly higher among amicrofilaremic than among microfilaremic individuals, and it was significantly higher in younger than in older persons. The highest mean IgG4 level was seen in young microfilaremic children, where the level was significantly higher than that in amicrofilaremic children of the same age group and that of older individuals irrespective of mf status. For those 10 years of age and older, the difference in mean level of IgG4 between microfilaremic and amicrofilaremic individuals was not significant. The prevalence of positivity for circulating antigens was 28% in the 1-4-year-old age group, and it increased gradually to 84% in the 50-59-year-old age group (average of 55% for all examined). When analyzed in relation to circulating antigen status, the difference in antibody levels between microfilaremic and amicrofilaremic adults decreased for IgG1 but increased for IgG4, indicating that the IgG1 levels were more related to mf status than to infection status, whereas the IgG4 levels were more related to infection status than to mf status.

High proportion of subclinical Plasmodium falciparum infections in an area of seasonal and unstable malaria in Sudan.

In the present longitudinal study, a cohort (n = 98) of children and adults 5-30 years of age living in an area of highly seasonal and unstable malaria transmission were followed for malaria morbidity during several successive transmission seasons. Based on morbidity surveillance during 1993 and measurements of antibody titers to the Plasmodium falciparum ring-infected erythrocyte surface antigen (Pf155/RESA), the cohort was divided into three groups: those who had at least one episode of clinical malaria (Group 1, n = 31), those who did not suffer from clinical malaria but had (Group 2, n = 63) or had not (Group 3, n = 4) a significant increase in antibody titers against the Pf155/RESA antigen. This increase was defined as equal to or greater than a four-fold increase in antibody titer in samples from same individuals taken at the beginning and the end of the malaria transmission season. Such increases in specific antibody levels suggested that the donors had been exposed to a P. falciparum blood-stage infection. Measurements of antibody titers to a peptide derived from the glutamate-rich protein exoantigen gave data parallel to those for Pf155/RESA. A surprisingly high fraction of individuals in the study cohort (approximately 66%) showed evidence of infection without ensuing clinical disease (Group 2).

Distinguishing Plasmodium falciparum treatment failures from re-infections by using polymerase chain reaction genotyping in a holoendemic area in northeastern Tanzania.

An in vivo drug sensitivity study was conducted in Magoda village in northeastern Tanzania to evaluate the usefulness of polymerase chain reaction (PCR)-based genotyping of Plasmodium falciparum parasites to distinguish between re-infection and treatment failure. The study tested P. falciparum susceptibility to a combination of sulfadoxine/pyrimethamine (Fansidar; F. Hoffmann La Roche, Basel, Switzerland). Blood samples were collected before treatment and on days 7, 14, or 28 post-treatment in 51 asymptomatic children, of which 26 could not clear parasitemia within seven days post-treatment. Among the remaining 25 children who had no detectable parasites on day 7, only five remained parasite negative up to day 28. Primary and recrudescent P. falciparum parasites were analyzed by PCR using family specific primers for merozoite surface protein-1 (MSP-1), MSP-2, and glutamate-rich protein (GLURP). All samples contained multiple P. falciparum infections. For all children with recrudescent P. falciparum, common alleles were detected in both the primary and recrudescent samples. However, in no child were the exact same alleles detected in both samples, indicating that probably at least some of the recrudescing parasites originated from new infections. The study demonstrates the general usefulness of PCR genotyping technique in distinguishing re-infections from true recrudescences following therapeutic drug treatment.

Suppressed peripheral and placental blood lymphoproliferative responses in first pregnancies: relevance to malaria.

An understanding of processes that predispose pregnant women, and in particular primigravidae, to malaria infection is essential to improve malaria management in pregnancy. Lymphoproliferative responses to malaria-specific (F32, 190L, and 190N) as well as other antigens (Candida and purified protein derivative [PPD]) were examined in the peripheral and placental blood of 102 Gambian women at the time of delivery. The lymphoproliferative responses of placental cells were poor to all antigens compared with those of peripheral blood (Candida P < 0.001, PPD P < 0.001, F32 P = 0.008, 190L P = 0.003, and 190N P = 0.10). Reduced proliferative capacity of placental mononuclear cells may contribute to heavy parasite colonization of this organ. Proliferation to malarial and PPD but not Candida antigens was selectively suppressed in peripheral and placental blood of primiparae relative to multiparae (F32 P = 0.07, 190L P = 0.09, 190N P = 0.007, PPD P = 0.09). Autologous plasma contained factors that suppressed lymphoproliferative responses to the same series of antigens to which the primiparae responded poorly (F32 P < 0.001, 190L P < 0.001, 190N P < 0.001, PPD P = 0.03). Malarial antibody levels were comparable among women of different parities and between peripheral and placental blood. Primigravidae may be more susceptible to malaria because of unique physiologic factors, such as higher levels of circulating immunosuppressive corticosteroids (P < 0.001), rather than differences in levels of acquired immunity.

Evidence of endothelial inflammation, T cell activation, and T cell reallocation in uncomplicated Plasmodium falciparum malaria.

To explain the observation that acute Plasmodium falciparum malaria is associated with a transient inability of peripheral blood cells to respond to antigenic stimulation in vitro, we have postulated the disease-induced reallocation of peripheral lymphocytes, possibly by adhesion to inflamed endothelium. We measured plasma levels of soluble markers of endothelial inflammation and T cell activation in 32 patients suffering from acute, uncomplication P. falciparum malaria, as well as in 10 healthy, aparasitemic control donors. All donors were residents of a malaria-endemic area of Eastern State Sudan. In addition, we measured the T cell surface expression of the interleukin-2 receptor (CD25) and the lymphocyte function-associated antigen (LFA-1; CD11a/CD18). We found that the plasma levels of all inflammation and activation markers were significantly increased in the malaria patients compared with the control donors. In addition, we found a disease-induced depletion of T cells with high expression of the LFA-1 antigen, particularly in the CD4+ subset. The results obtained provide further support for the hypothesis of T cell reallocation to inflamed endothelium in acute P. falciparum malaria.

Immunoglobulin G reactivities to rhoptry-associated protein-1 associated with decreased levels of Plasmodium falciparum parasitemia in Tanzanian children.

In the Muheza region of Tanzania, an area with holoendemic malaria, the proportion of responders with IgG enzyme-linked immunosorbent assay reactivities to recombinant rhoptry-associated protein-1 (rRAP-1) as well as IgG reactivities to a repeat region of the acidic-basic repeat antigen (ABRA) increased with age. The proportion of responders with IgM reactivities to rRAP-1 increased with age in the first three decades. However, levels of IgG reactivities to rRAP-1 did not increase with age, indicating high levels of reactivities among young children. High P. falciparum densities were only detectable in children less than five years of age; in this group the proportion of IgG responders to rRAP-1 and to the ABRA repeat region was low but levels of IgG reactivities to rRAP-1 were inversely correlated with parasite density, suggesting that immune recognition of the antigen may be associated with resistance to infection. On the other hand, levels of IgG reactivities to the repeat region of ABRA increased with parasite densities in children 1-4 years of age. Two different profiles of IgG reactivities to rRAP-1 and to ABRA are detectable in young Tanzanian children and the Ig reactivities against rRAP-1 may be a component of the immune reactions restricting parasite multiplication.

A prospective study of the influence of alpha thalassaemia on morbidity from malaria and immune responses to defined Plasmodium falciparum antigens in Gambian children.

The protective effect of alpha thalassaemia (-alpha/alpha alpha) against morbidity from falciparum malaria was assessed in a prospective study of rural Gambian children. The gene frequency for single alpha-globin gene deletions was 0.12. Malariometric indices measured during cross-sectional surveys and morbidity from malaria determined by weekly surveillance were similar in children with alpha thalassaemia and in those with a normal alpha-globin genotype. However, the small number of children who carried both alpha thalassaemia and the sickle cell trait had fewer clinical episodes of malaria than children with the sickle cell trait alone. Specific antibody responses and cell-mediated immune responses in vitro to defined Plasmodium falciparum antigens were measured in children participating in the study. In general, there was no evidence of an increased prevalence or intensity of humoral or cell-mediated immune responses to the malaria antigens studied in children heterozygous for alpha thalassaemia compared with children with a normal alpha-globin genotype.

Maloprim malaria prophylaxis in children living in a holoendemic village in north-eastern Tanzania.

A randomized, double-'blind', placebo-controlled trial of weekly Maloprim (dapsone-pyrimethamine, D-P) for malaria prophylaxis was conducted at Magoda village in north-eastern Tanzania. The effect of D-P on the incidence of clinical malaria, Plasmodium falciparum prevalence and density, splenomegaly, and packed cell volume (PCV) was investigated in a cohort of 249 children (126 receiving D-P and 123 receiving placebo) aged 1-9 years. The case definition of clinical malaria (malaria fever) was measured axillary temperature > or = 37.5 degrees C and/or reported fever, and P. falciparum asexual parasitaemia > or = 5000/microL. Children aged 1-4 years given D-P experienced 1.56 episodes of clinical malaria per year, whereas children on placebo experienced 2.55 episodes (relative rate [RR] = 0.61, 95% confidence interval [CI] 0.47, 0.80). Thus, D-P protective efficacy against clinical malaria, in this age group, was 39% (95% CI 20%, 53%; P = 0.0002). The annual incidence of clinical malaria among children aged 5-9 years was 0.16 episodes in the D-P group and 0.26 episodes in those receiving placebo (RR = 0.58, 95% CI 0.26, 1.28; P = 0.17). Increased malaria transmission and drug resistance, during the course of the trial, resulted in a reduction in the protective efficacy of D-P. Overall, D-P was able to reduce parasite densities and splenomegaly. D-P prophylaxis also resulted in an increase in PCV but this effect diminished towards the end of the trial. D-P exerted a suppressive effect on asexual parasitaemia throughout the trial.

Differential antibody response of Gambian donors to soluble Plasmodium falciparum antigens.

A seroepidemiological and clinical study was performed in an area of West Africa (The Gambia) where Plasmodium falciparum is endemic with seasonal transmission. Plasma samples were tested by intermediate gel immunoelectrophoresis for antibodies against 7 soluble P. falciparum antigens. There were marked differences in the age-related pattern of antibody response to the different antigens. Antibodies to 4 of the antigens were acquired slowly with a maximum prevalence reached after 25-35 years of age. Antibodies against the 3 remaining antigens, including the endotoxin-like antigen, Ag7, were acquired earlier with a plateau of maximum prevalence reached after 5-11 years, i.e. at the time when morbidity due to malaria decreased. Children who had not appeared to be infected with malaria during the preceding transmission season had lower levels of antibodies to soluble antigens than did children who had had a documented attack of clinical malaria or parasitaemia. There was no difference in antibody profiles to soluble antigens between children with sickle cell trait and children with normal haemoglobin.