Correlates of HIV and malaria co-infection in Southern India

ABSTRACT: BACKGROUND: Malaria and HIV co-infection adversely impact the outcome of both diseases and previous studies have mostly focused on falciparum malaria. Plasmodium vivax contributes to almost half of the malaria cases in India, but the disease burden of HIV and P. vivax co-infection is unclear. METHODS: HIV-infected subjects (n=460) were randomly selected from the 4,611 individuals seen at a Voluntary Counseling and Testing Center in Chennai, India between Jan 2 to Dec 31 2008. Malaria testing was performed on stored plasma samples by both nested PCR using both genus-specific and species-specific primers and immunochromatography-based rapid diagnostic test for detecting antibodies against both Plasmodium falciparum and P. vivax. RESULTS: Recent malaria co-infection, defined by the presence of antibodies, was detected in 9.8% (45/460) participants. Plasmodium vivax accounted for majority of the infections (60%) followed by P. falciparum (27%) and mixed infections (13%). Individuals with HIV and malaria co-infection were more likely to be men (p=0.01). Between those with and without malaria, there was no difference in age (p=0.14), CD4+ T-cell counts (p=0.19) or proportion CD4+ T-cell below 200/mL (p=0.51). CONCLUSIONS: Retrospective testing of stored plasma samples for malaria antibodies can facilitate identification of populations with high rates of co-infection, and in this southern India HIVinfected cohort there was a considerable burden of malaria co-infection, predominantly due to P. vivax. However, the rate of P. falciparum infection was more than 6-fold higher among HIV-infected individuals than what would be expected in the general population in the region. Interestingly, individuals co-infected with malaria and HIV were not more likely to be immunosuppressed than individuals with HIV infection alone.     

Naturally acquired CD8+ cytotoxic T lymphocytes against the Plasmodium falciparum circumsporozoite protein.

In rodent malaria model systems, protective immunity induced by immunization with irradiated sporozoites is eliminated by in vivo depletion of CD8+ T cells, and adoptive transfer of CTL clones against the circumsporozoite protein protects against malaria. We recently demonstrated that volunteers immunized with irradiated Plasmodium falciparum sporozoites produce CTL against peptide 368-390 of the P. falciparum circumsporozoite protein. To determine whether natural exposure to malaria induced similar CTL, we studied 11 adult, male, life-long residents of a highly malarious area of Kenya, who were selected because their lymphocytes had been shown to proliferate after stimulation with peptides 361-380, 371-390, or 368-390 and because nine had been resistant to malaria in previous studies. In four of the 11 individuals there was peptide-specific, genetically restricted, CTL activity. In all four individuals, this activity was unaffected by depletion of CD4+ T cells. In three volunteers the activity was eliminated or reduced by depletion of CD8+ T cells; in the fourth volunteer the CD8+ T cell depletion was uninterpretable. This first demonstration of CD8+ T cell, genetically restricted, Ag-specific CTL against a malaria protein among individuals exposed to endemic malaria provides a foundation for studying the relationship between circulating CTL and resistance to malaria infection.     

Human CD4+ T cells induced by synthetic peptide malaria vaccine are comparable to cells elicited by attenuated Plasmodium falciparum sporozoites

Peptide vaccines containing minimal epitopes of protective Ags provide the advantages of low cost, safety, and stability while focusing host responses on relevant targets of protective immunity. However, the limited complexity of malaria peptide vaccines raises questions regarding their equivalence to immune responses elicited by the irradiated sporozoite vaccine, the "gold standard" for protective immunity. A panel of CD4+ T cell clones was derived from volunteers immunized with a peptide vaccine containing minimal T and B cell epitopes of the Plasmodium falciparum circumsporozoite protein to compare these with previously defined CD4+ T cell clones from volunteers immunized with irradiated P. falciparum sporozoites. As found following sporozoite immunization, the majority of clones from the peptide-immunized volunteers recognized the T* epitope, a predicted universal T cell epitope, in the context of multiple HLA DR and DQ molecules. Peptide-induced T cell clones were of the Th0 subset, secreting high levels of IFN-gamma as well as variable levels of Th2-type cytokines (IL-4, IL-6). The T* epitope overlaps a polymorphic region of the circumsporozoite protein and strain cross-reactivity of the peptide-induced clones correlated with recognition of core epitopes overlapping the conserved regions of the T* epitope. Importantly, as found following sporozoite immunization, long-lived CD4+ memory cells specific for the T* epitope were detectable 10 mo after peptide immunization. These studies demonstrate that malaria peptides containing minimal epitopes can elicit human CD4+ T cells with fine specificity and potential effector function comparable to those elicited by attenuated P. falciparum sporozoites.     

Antigen-specific and MHC-restricted Plasmodium falciparum-induced human T lymphocyte clones

We established and analyzed human T lymphocyte clones induced by crude Plasmodium falciparum antigens of schizont-enriched asexual blood stages. Peripheral blood mononuclear cells (PBMC) were stimulated for 6 days with antigen, and the T cell blasts were separated and were transferred to limiting dilution cultures with antigen, irradiated PBMC, and recombinant interleukin 2. The following observations were made. Malaria antigen (M.Ag) induced similar proportions of T blasts in PBMC from infected individuals and noninfected controls, and the M.Ag-dependent clone frequencies (1/79 to 1/216) obtained with the blasts were similar. The majority of established clones derived from infected and noninfected subjects specifically recognized M.Ag and would not proliferate in response to red blood cells or autologous PBMC alone. They also required HLA class II determinant-compatible antigen-presenting (E-) cells. With three clones from one malaria patient, DR 1 or DR 5 specificities correlated with antigen presentation. Although T4+ and T8+ blasts were induced by M.Ag in PBMC, only T4 (Leu-3+) clones were obtained in our culture system. These clones secreted IL 2 in response to M.Ag. 4) Differential patterns of reactivity to native M.Ag, heat-stable antigens, and heat-precipitated antigens were exhibited by T cell clones, and the tested clones did not recognize Plasmodium berghei antigen. In conclusion, it is important with regard to previous observations on apparently nonspecific, mitogen-like effects of M.Ag in bulk T cell cultures that our results demonstrate specific recognition of P. falciparum by human T cells. The T cell clones obtained will be an important tool in the quest for a better understanding of the mechanisms involved in resistance to malaria infection.     

Cross-reactive anti-PfCLAG9 antibodies in the sera of asymptomatic parasite carriers of Plasmodium vivax

The PfCLAG9 has been extensively studied because their immunogenicity. Thereby, the gene product is important for therapeutics interventions and a potential vaccine candidate. Antibodies against synthetic peptides corresponding to selected sequences of the Plasmodium falciparum antigen PfCLAG9 were found in sera of falciparum malaria patients from Rondônia, in the Brazilian Amazon. Much higher antibody titres were found in semi-immune and immune asymptomatic parasite carriers than in subjects suffering clinical infections, corroborating original findings in Papua Guinea. However, sera of Plasmodium vivax patients from the same Amazon area, in particular from asymptomatic vivax parasite carriers, reacted strongly with the same peptides. Bioinformatic analyses revealed regions of similarity between P. falciparum Pfclag9 and the P. vivax ortholog Pvclag7. Indirect fluorescent microscopy analysis showed that antibodies against PfCLAG9 peptides elicited in BALB/c mice react with human red blood cells (RBCs) infected with both P. falciparum and P. vivax parasites. The patterns of reactivity on the surface of the parasitised RBCs are very similar. The present observations support previous findings that PfCLAG9 may be a target of protective immune responses and raises the possibility that the cross reactive antibodies to PvCLAG7 in mixed infections play a role in regulate the fate of Plasmodium mixed infections.     

Effect of sequential infection with Plasmodium vivax and P. falciparum in the Aotus trivirgatus monkey.

Aotus trivirgatus monkeys with prior experience with Plasmodium vivax were inoculated with P. falciparum via the bites of infected mosquitoes. The animals with prior malaria had higher parasitemias and significantly higher levels of mosquito infectivity than monkeys with no prior P. vivax experience. Monkeys with a history of P. falciparum that were inoculated with P. vivax had essentially the same parasitemias as those with no prior malaria. However, levels of mosquito infectivity were markedly increased in those monkeys with a history of P. falciparum. The results imply that the introduction of another malaria species into a malarious area may result in higher levels of mosquito infection and more rapid establishment and distribution of that species.     

Severe disease in children hospitalized with a diagnosis of Plasmodium vivax in south-eastern Pakistan

ABSTRACT: BACKGROUND: Infection by Plasmodium vivax has been considered rarely threatening to life, but recent studies challenge this notion. This study documented the frequency and character of severe illness in paediatric patients admitted to a hospital in south-eastern Pakistan with a laboratory-confirmed diagnosis of vivax malaria. METHODS: An observational study of all 180 paediatric patients admitted with any diagnosis of malaria during 2010 was conducted: 128 P. vivax; 48 Plasmodium falciparum; and four mixed infections of these species. Patients were classified as having severe illness with any of the following indicators: Glascow coma scale <11; >2 convulsions; haemoglobin <5g/dL; thrombocytes <50,000/mL; blood glucose <45mg%; >70 breaths/min; or intravenous antimalarial therapy. Additionally, 64 patients with a diagnosis of vivax malaria were treated during 2009, and the 21 of these having severe illness were included in analyses of the frequency and character of severe illness with that diagnosis. RESULTS: During 2010, 39 (31%) or 37 (77%) patients with a diagnosis of P. vivax or P. falciparum were classified as having severe disease. Including the 2009 records of 64 patients having vivax malaria, a total of 60 (31%) patients with severe illness and a diagnosis of P. vivax were available. Altered mental status (Glascow coma scale score <11; or >2 convulsions) dominated at 54% of the 83 indicators of severe illness manifest among the patients with vivax malaria, as was true among the 37 children with a diagnosis of falciparum malaria and being severely ill; 58% of the 72 indicators of severe disease documented among them. No statistically significant difference appeared in frequencies of any other severe disease indicators between patients diagnosed with vivax or falciparum malaria. Despite such similarities, a diagnosis of falciparum malaria nonetheless came with 3.8-fold (95% CI = 1.8- 8.1) higher risk of presenting with severe illness, and 8.0-fold (95% CI = 2.1-31) greater likelihood of presenting with three or more severe disease indicators. Two patients did not survive hospitalization, one each with a diagnosis of falciparum or vivax malaria. CONCLUSIONS: Vivax malaria caused a substantial burden of potentially life-threatening morbidity on a paediatric ward in a hospital in south-eastern Pakistan.     

T cell epitopes of the circumsporozoite protein of Plasmodium vivax. Recognition by lymphocytes of a sporozoite-immunized chimpanzee.

The humoral and cellular antisporozoite immune responses of a laboratory-born chimpanzee were measured following multiple exposures to the bites of Plasmodium vivax-infected mosquitoes. T cell lines and clones derived from the chimpanzee's PBL were used to identify T cell epitopes of the P. vivax circumsporozoite (CS) protein. Two independently obtained cell lines, established by culturing the PBL with either a recombinant P. vivax circumsporozoite (rPvCS) protein or a pool of synthetic peptides spanning the rPvCS sequence, recognized a 20-mer peptide from a nonpolymorphic region of the carboxyl terminus of the CS protein. This peptide overlaps a sequence homologous to region II of the Plasmodium falciparum CS protein. A third T cell line recognized an epitope within the central repeat domain, which has recently been found to be a polymorphic region of the P. vivax CS protein. The CD4+ clones derived from this third T cell line secreted IFN-gamma and IL-2 when stimulated with either the P. vivax repeat peptide (DRAAGQPAG)2 or the rPvCS protein.     

Human T cells recognize polymorphic and non-polymorphic regions of the Plasmodium falciparum circumsporozoite protein.

In order to characterize T cell epitopes in the Plasmodium falciparum circumsporozoite (CS) protein sequence, we isolated T cell clones, from non-immune donors, which reacted with synthetic peptides corresponding to two predicted CS protein T cell epitopes. Peptide CS.T3 (corresponding to a non-polymorphic region of the CS protein, residues 378-398) was recognized in association with either DR2 or DRw9 restriction elements. T cell clones recognizing CS.T3 also reacted with the sporozoite-derived CS protein. Peptide CS.T2 corresponds to a polymorphic region (residues 325-341) of the CS protein. Unlike the CS.T3-specific clones, the CS.T2-specific clones did not recognize the CS protein. Since the CS.T2 peptide includes residues which are polymorphic in different P. falciparum isolates, we investigated whether these residues were critical for recognition of the peptide. We show here that a single amino acid substitution at a position of the CS protein which shows genetic polymorphism affects recognition of the sequence by human T cells. The implications of these data for malaria vaccine development are discussed.     

Prevalence and level of antibodies to the circumsporozoite protein of human malaria parasites in five states of the Amazon region of Brazil

The aim of this study was to determine the prevalence of malaria infection and antibodies against the repetitive epitopes of the circumsporozoite (CS) proteins of Plasmodium falciparum, P. malariae, P. vivax VK210, P. vivax VK247, and P. vivax-like in individuals living in the states of Rond?nia, Pará, Mato Grosso, Amazonas, and Acre. Active malaria transmission was occurring in all studied sites, except in Acre. P. falciparum was the predominant species in Pará and Rond?nia and P. vivax in Mato Grosso. Infection by P. malariae was low but this Plasmodium species was detected in Rond?nia (3.5%), Mato Grosso (2.5%), and Pará (0.8%). High prevalence and levels of serological reactivity against the CS repeat peptides of P. falciparum were detected in Rond?nia (93%) and Pará (85%). Sera containing antibodies against the CS repeat of P. malariae occurred more frequently in Rond?nia (79%), Pará (76%), and Amazonas (68%). Antibodies against the repeat epitope of the standard CS protein of P. vivax VK210, P. vivax VK247, and P. vivax-like were more frequent in Rond?nia, Pará, and Mato Grosso. The high frequency of reactions to P. malariae in most of the areas suggests that the infection by this Plasmodium species has been underestimated in Brazil.     

Heterogeneity among human T cell clones recognizing an HLA-DR4,Dw4-restricted epitope from the 18-kDa antigen of Mycobacterium leprae defined by synthetic peptides.

Synthetic peptides have been used to exactly define a T cell epitope region from the immunogenic 18-kDa protein of Mycobacterium leprae. Four M. leprae reactive CD4+ T cell clones, isolated from two healthy individuals vaccinated with killed M. leprae, recognized a determinant initially defined by the peptide (38-50). However, fine mapping of the minimal sequence required for T cell recognition revealed heterogeneity among the T cell clones with regard to the N- and carboxyl-terminal boundaries of the epitopes recognized. MHC restriction analysis showed that the immunogenic peptides were presented to the T cells in an HLA-DR4,Dw4-restricted manner in all cases. The results suggest that a polyclonal T cell response representing different fine specificities is directed toward a possible immunodominant epitope from the M. leprae 18-kDa Ag in individuals carrying this MHC haplotype.     

Plasmodium falciparum and P. vivax gametocyte-specific exoantigens stimulate proliferation of TCR gammadelta+ lymphocytes

Immune modulation of Plasmodium vivax and P. falciparum gametocytes occurs over the course of erythrocytic infection. The response is linked to proliferative and inflammatory responses, which may be stimulated by stage-specific gametocyte proteins. Stage-specific exoantigens were purified from supernatants of P. falciparum and P. vivax gametocyte cultures, and either primary or secondary postinfection lymphocytes were stimulated for proliferation. Five of 25 exoantigens purified from P. falciparum gametocyte cultures and 6 of 28 exoantigens isolated from P. vivax were gametocyte stage specific. Metabolic labeling of soluble P. falciparum gametocyte proteins confirmed synthesis and secretion of 5 stage-specific exoantigens, with molecular masses of 118, 62, 52, 37, and 33 kDa. Purified gametocyte exoantigens within the range of 50 to 100 kDa stage-specifically stimulated proliferation of lymphocytes from postprimary P. falciparum infections, and from postprimary and secondary P. vivax infection patients with homologous purified exoantigens. T-cell receptor (TCR)gammadelta+, and CD3+ CD8+ and CD3+ CD4- CD8- T cells were specifically upregulated from P. falciparum primary- and P. vivax secondary-infection lymphocytes, respectively, using gametocyte stage-specific exoantigens. CD25+ was the major activation marker expressed by CD3+ and gammadelta T cells when stimulated with gametocyte exoantigens. None of the T cell markers was significantly upregulated using gametocyte stage-specific exoantigens with primary-infection P. vivax lymphocytes.     

Frequency of cytokine-producing CD4-CD8- peripheral blood mononuclear cells in patients with Plasmodium falciparum malaria.

The frequency of cytokine-producing CD4-/CD8- mononuclear cells was assessed in patients of different age groups (29 infants, aged 1-5 years; 30 schoolchildren, aged 6-14 years, 26 adults, aged > 15 years) with acute Plasmodium falciparum malaria, from Gabon. Fifteen patients were followed up before antimalarial treatment (day 0), during parasite clearance (day 3) and after resolution of parasitemia (day 10). By using flow cytometry for intracellular detection of cytokines, a striking expansion of CD4-/CD8- cells producing the type 1 cytokines interleukin (IL)-2-/interferon (IFN)-gamma+, IL-2+/IFN-gamma+ and IL-2+/IFN-gamma- was observed in adults as compared with children. Type 2 cytokine expression (IL-4+/IFN-gamma-, IL-13+/IFN-gamma-) and type 0 cells (IL-4+/IFN-gamma+, IL-13+/IFN-gamma+) were not significantly different between the three age groups. Patients with severe malaria had a significantly increased frequency of type 2 cytokine-producing CD4-/CD8- cells. Drug-induced clearance of parasitemia was characterized by a decrease of IL-2+/IFN-gamma- and type 2 cytokine expressing CD4-/CD8- cells and by a gradual increase of IL-10+/IFN-gamma- expression. The type 1/type 2 dichotomy observed within the CD4-/CD8- cell population is likely to be of significance in the host response against P. falciparum malaria.     

Human T-cell responses to malaria: mostly forgotten or committed to memory?

T cells have been implicated in both malaria immunity and malaria disease and factors controlling the maintenance of T-cell responses over time may alter the clinical outcome o f infection. Michael Good and Janine Bilsborough have compared the T-cell responses to epitopes from the Plasmodium vivax and P. falciparum circumsporozoite proteins and here discuss the issue of T-cell memory as it applies to malaria.     

An update on the search for a Plasmodium vivax vaccine

Although Plasmodium falciparum is the leading cause of morbidity and mortality due to malaria worldwide, nearly 2.5 billion people, mostly outside Africa, are also at risk from malaria caused by Plasmodium vivax infection. Currently, almost all efforts to develop a malaria vaccine have focused on P. falciparum. For example, there are 23 P. falciparum vaccine candidates undergoing advanced clinical studies and only two P. vivax vaccine candidates being tested in preliminary (Phase I) clinical trials, with few others being assessed in preclinical studies. More investment and a greater effort toward the development of P. vivax vaccine components for a multi-species vaccine are required. This is mainly because of the wide geographical coexistence of both parasite species but also because of increasing drug resistance, recent observations of severe and lethal P. vivax cases and relapsing parasite behaviour. Availability of the P. vivax genome has contributed to antigen discovery but new means to test vaccines in future trials remain to be designed.     

A Plasmodium falciparum copper-binding membrane protein with copper transport motifs

Formerly known as a hypoendemic malaria country, the Republic of Djibouti declared the goal of pre-eliminating malaria in 2006. The aim of the present study was to evaluate the prevalence of Plasmodium falciparum, Plasmodium vivax and mixed infections in the Djiboutian population by using serological tools and to identify potential determinants of the disease and hotspots of malaria transmission within the country. The prevalence of P. falciparum and P. vivax within the districts of the capital city and the rest of the Republic of Djibouti were assessed using 13 and 2 serological markers, respectively. The relationship between the immune humeral response to P. falciparum and P. vivax and variables such as age, gender, wealth status, urbanism, educational level, distance to rivers/lakes, living area, having fever in the last month, and staying in a malaria-endemic country more than one year was estimated and analysed by questionnaires administered to 1910 Djiboutians. Multivariate ordinal logistic regression models of the immune humeral response were obtained for P. falciparum and P. vivax. The P. falciparum and P. vivax seroprevalence rates were 31.5%, CI95% [29.4-33.7] and 17.5%, CI95% [15.8-19.3], respectively. Protective effects against P. falciparum and P. vivax were female gender, educational level, and never having visited a malaria-endemic area for more than one year. For P. falciparum only, a protective effect was observed for not having a fever in the last month, living more than 1.5 km away from lakes and rivers, and younger ages. This is the first study that assessed the seroprevalence of P. vivax in the Republic of Djibouti. It is necessary to improve knowledge of this pathogen in order to create an effective elimination programme. As supported by recent observations on the subject, the Republic of Djibouti has probably demonstrated a real decrease in the transmission of P. falciparum in the past seven years, which should encourage authorities to improve efforts toward elimination.     

Major histocompatibility complex and T cell interactions of a universal T cell epitope from Plasmodium falciparum circumsporozoite protein

A 20-residue sequence from the C-terminal region of the circumsporozoite protein of the malaria parasite Plasmodium falciparum is considered a universal helper T cell epitope because it is immunogenic in individuals of many major histocompatibility complex (MHC) haplotypes. Subunit vaccines containing T* and the major B cell epitope of the circumsporozoite protein induce high antibody titers to the malaria parasite and significant T cell responses in humans. In this study we have evaluated the specificity of the T* sequence with regard to its binding to the human class II MHC protein DR4 (HLA-DRB1*0401), its interactions with antigen receptors on T cells, and the effect of natural variants of this sequence on its immunogenicity. Computational approaches identified multiple potential DR4-binding epitopes within T*, and experimental binding studies confirmed the following two tight binding epitopes: one located toward the N terminus (the T*-1 epitope) and one at the C terminus (the T*-5 epitope). Immunization of a human DR4 volunteer with a peptide-based vaccine containing the T* sequence elicited CD4+ T cells that recognize each of these epitopes. Here we present an analysis of the immunodominant N-terminal epitope T*-1. T*-1 residues important for interaction with DR4 and with antigen receptors on T*-specific T cells were mapped. MHC tetramers carrying DR4/T*-1 MHC-peptide complexes stained and efficiently stimulated these cells in vitro. T*-1 overlaps a region of the protein that has been described as highly polymorphic; however, the particular T*-1 residues required for anchoring to DR4 were highly conserved in Plasmodium sequences described to date.