HIV infection is an increasing health problem in sub-Saharan Africa where malaria is endemic, and these two diseases are serious threats to public health.1,2 Like malaria parasites, HIV is an intracellular pathogen. Malaria parasites reside in the hepatocytes as the pre-erythrocytic stages and then in the red blood cells as the erythrocytic stages, while HIV mostly attacks CD4 cells and macrophages. It is well appreciated that cell-mediated immunity (CMI) plays an important protective role against both HIV and acute malaria infections.3,4
Because HIV infections result in generalized immunosuppression by depleting CD4+ T lymphocytes, HIV/AIDS can have a broad impact on the ability of the immune system of HIV-infected patients to control opportunistic infections and other infectious diseases. Previous studies have demonstrated that malaria infection is exacerbated in HIV-positive patients. Whitworth et al reported that the level of parasitemia and clinical episodes due to malaria were significantly increased in HIV-positive adults patients in Uganda.5 Another study in Uganda demonstrated that HIV-positive children had more-severe clinical outcomes than those who were HIV-negative.6 In contrast, other studies have found no observable interaction between malaria and HIV infection. Studies in Kinshasa demonstrated that the clinical outcome of malaria was the same for both HIV-positive and HIV-negative children, and that there was no difference in malaria parasitemia between HIV-positive and HIV-negative children.7,8 Another study in Kigali demonstrated that malaria parasitemia in pregnant HIV-positive women was the same as in pregnant HIV-negative women.9 Recent studies on patients in Malawi10,11 have reported increased HIV-1 viral load and reversible induction of HIV-1 replication in CD14 macrophages during acute malaria infection caused by Plasmodium falciparum.
Based on the fact that healthy people living in malaria endemic areas who are exposed to repeated malaria infections can develop a partially protective non-sterilizing immunity against the infection, we hypothesized that a disruption of the immune system as commonly observed in HIV patients may result in quantitative and/or qualitative differences in antibody against malaria parasites, thus affecting the outcome of malaria infection. To investigate the possible interactions between HIV and malaria infections, 47 specimens from 25 HIV-discordant and -concordant couples (mean age 39; median age, 41; age range, 26–51) who had been selected during a pilot study in a large factory in Kinshasa in 1998 and who lived in P. falciparum malaria endemic areas were analyzed. The primary purpose of the study was to assess the HIV cytotoxic T lymphocytes response in both the HIV+ subject and the subject’s HIV− partner. Lack of availability of flow cytometry facility in Kinshasa and poor viability of transported cells prevented a direct measurement of CD4 T cell count. What is known, however, is that these couples living in malaria endemic area had experienced numerous episodes of clinical malaria in their lifetimes. According to the national malaria control program of the DRC, nearly 44% of outpatients of all ages at representative hospitals in Kinshasa had a clinical diagnosis of malaria from 1997–1999. Moreover, the higher incidence of malaria in Kinshasa occurs from June–July and December–January. The samples analyzed in our study were collected from May 1998 to January 1999 with an interruption from August to November because of war in Kinshasa. The studies reported here were approved by an ethics committee in the DRC and the Joint Committee on Clinical Investigation of Johns Hopkins University.
HIV viral load was estimated by determining HIV-1 RNA levels in the plasma by the reverse transcriptase-polymerase chain reaction method (Amplicor HIV-1 Monitor Assay, version 1.5, Roche Molecular Diagnostics, Branchburg, NJ). The level of sensitivity of the assay is 50 copies/mL. A standard enzyme-linked immunosorbent assay (ELISA) was used to measure antibodies recognizing antigens in asexual erythrocytic stage P. falciparum.12 Cultured P. falciparum13 (trophozoite and schizont stages, 3D7 strain) were lysed by sonication, and the supernatant obtained after centrifugation at 14,000 rpm (30 min, 4°C) was used to coat ELISA plates (1 μg protein/well in bicarbonate buffer). After blocking with 5% non-fat milk, the plates were washed twice with phosphate-buffered saline-0.1% Tween 20 and incubated with various serial dilutions of plasma from the study participants and a pool of 10 North American normal human sera (negative controls) for one hour at 37°C. After further washing, 100 μL/well of peroxidase-conjugated goat anti-human IgM and IgG antibodies diluted in blocking solution (1:500) was added, and the plates incubated for one hour at 37°. After a final wash, 100 μL/well of chromogen containing substrate (ABTS) mixture was added, and reactions were stopped after 45 minutes. Optical densities were measured using a plate reader at 450 nm.
Anti-P. falciparum antibodies and HIV status.
To investigate the relationship between malaria and HIV, malaria antibody titers were compared in 23 HIV-positive (12 female) and 24 HIV-negative individuals (14 male). A χ2 analysis showed no correlation between antibodies against malaria parasite and HIV status (P = 0.370). Table 1 represents an overall summary of specimens, gender, results of malaria antibody titer, HIV western blot results, viral load, and results on malaria growth inhibition assays. In this analysis, we compared antibody titers as well as simple antibody-positive or -negative data. While HIV western blot identifies HIV infection, a more-effective gauge of the clinical status of an HIV-infected individual is the viral load. Data were entered into Excel worksheets to analyze for significance of correlation. Statistical analysis and χ2 and t tests were done using the Sigma Stat 5 software program. Table 2 summarizes the results of various statistical analyses performed. No correlation was observed among the test parameters used for comparison. Further investigation did not find any significant relationship between gender and other parameters used in the study: HIV status (P = 0.876), viral load (P = 0.412), malaria antibody titer (P = 0.141), and malaria ELISA (P = 0.319).
P. falciparum growth inhibition.
We then considered the possibility that similar concentrations of antibodies are produced in individuals regardless of HIV status but that they might differ with respect to antigen specificity and/or in their ability to inhibit parasite growth in the erythrocytes. To identify specific proteins recognized by various plasma samples, asexual cultures (3D7 parasites) were treated with 0.15% saponin and parasite proteins fractionated by SDS polyacrylamide gel electrophoresis 12% gel and transferred to nitrocellulose membrane for immunoblot analysis. Fifteen DRC plasma specimens (1:500 dilution) were used as the primary antibody and peroxidase-conjugated goat antihuman IgG (1: 5,000 dilution) as the secondary antibody with one hour of incubation for each step at room temperature. Membranes were washed four times between incubations with the primary and secondary antibodies, and detection was done using the ECL kit (Amersham Biosciences, Buckinghamshire). Among the 15 plasma specimens, six were HIV-positive and malaria antibody-positive, three were HIV-negative and malaria antibody-positive, three were HIV-positive and malaria antibody-negative, and three were HIV-negative and malaria antibody-negative. While there were sample-to-sample variations, analysis of banding pattern (5 to > 22 proteins recognized) failed to reveal any noticeable difference in the antigen recognition patterns among the various groups of samples for antibodies against P. falciparum irrespective of HIV status. (Data not shown.)
To evaluate parasite growth inhibitory activity, various DRC plasma and normal human sera were diluted in the culture medium and tested in P. falciparum in vitro growth inhibition assays. One hundred μL diluted plasma were added to each well containing 100 μL of P. falciparum culture (1% of parasitemia and 5% of hematocrit) in triplicate wells. The plates were incubated for 48 hours at 37°C (5% C02, 5% 02 and N2 balance) in modular chambers. Smears were made from each well, fixed by methanol, stained by Giemsa solution (5%), and examined by light microscopy using oil immersion lens (100x). Total parasites were counted in 10–15 randomly selected fields representing a minimum of 1,000 erythrocytes and percent parasitemia determined for each test group. The mean parasitemia in the control wells (normal human sera) was 3.8% after 48 hours. Although various plasma from the DRC had strong P. falciparum growth inhibition activity (Table 1), no significant correlation with HIV status was discernible (Table 2). In growth inhibition assay, 11 malaria antibody-negative sera also gave significant reduction of parasite growth, suggesting that besides antibodies, other unknown immunologic mediators may affect parasite growth and contribute to productive immunity. For example, previous studies have demonstrated an anti-parasite role for gamma interferon and other proinflammatory and anti-inflammatory cytokines.14,15
The pathogenesis of HIV infection is marked by the depletion of CD4+ T lymphocytes, an important component of CMI. HIV-associated immunosuppression may affect the prognosis of malaria by altering the immune response against P. falciparum, but such an interaction between malaria and HIV infection still remains highly debatable. Many clinical and in vitro studies have investigated such questions, and the results remain controversial. This issue led us to take advantage of samples from HIV-discordant and HIV-concordant couples living in malaria endemic areas and sharing a similar frequency of mosquito bites to approach this question. We found no significant correlation between HIV status and dysfunction of the immune response against malaria parasites. Our expectation was that altered immune responses during HIV infection will result in reduced antibodies against malaria antigens and thus negatively affecting the outcome of malaria during co-infection with HIV. In this cross-sectional study, we failed to observe any relationship between the various malaria parameters and HIV status. Knowledge of CD4+ and CD8+ cell number and evaluation of cell-mediated immune responses against various malaria antigens may provide a better understanding of immune interactions between HIV and malaria.
Summary of samples and results of malaria-specific antibody titers, HIV viral load, and percentage inhibition of parasite growth in vitro
| % Growth inhibition | |||||
|---|---|---|---|---|---|
| Couple | Gender | Malaria antibody titer* | HIV viral load** | 10% | 20% |
| Numbers in parentheses represent parameters for females. | |||||
| X: Result not available. | |||||
| *Reciprocal plasma dilution. | |||||
| **Similar results were obtained by HIV western blot analysis. | |||||
| *** Values represent percentage inhibition of Plasmodium falciparum growth in the presence of 10% or 20% DRC plasma. | |||||
| 1 | M (F) | 0 (1,600) | 10,719 (0) | 31 (45) | 52 (31) |
| 2 | M (F) | 800 (0) | 1,160 (0) | 41 (30) | 57 (25) |
| 3 | M (F) | 3,200 (25,600) | 7,306 (0) | 85 (53) | 94 (69) |
| 4 | M (F) | 0 (200) | 332,440 (27,489) | 31 (23) | 57 (51) |
| 5 | M (F) | 800 (12,800) | 0 (37,405) | 48 (52) | 50 (50) |
| 6 | M (F) | 1,600 (0) | 0 (1,632) | 33 (63) | 55 (65) |
| 7 | M (F) | 400 (0) | 18,876 (285,256) | 42 (71) | 69 (78) |
| 8 | M (F) | 1,600 (12,800) | 0 (1,287) | 36 (67) | 59 (76) |
| 9 | M (F) | 400 (400) | 0 (58,923) | 42 (83) | 58 (90) |
| 10 | M (F) | 3,200 (1,600) | 0 (26,899) | 57 (34) | 74 (44) |
| 11 | M (F) | 0 (0) | 0 (9,732) | 47 (47) | 70 (57) |
| 12 | M (F) | 200 (0) | 0 (62,592) | 50 (41) | 50 (71) |
| 13 | M (F) | 200 (400) | 0 (27,430) | 18 (52) | 54 (66) |
| 14 | M (F) | 1,600 (X) | 0 (X) | 28 (X) | 49 (X) |
| 15 | M (F) | 6,400 (200) | 24,749 (53,840) | 25 (22) | 48 (26) |
| 16 | M (F) | 200 (800) | 41,438 (0) | 39 (51) | 44 (33) |
| 17 | M (F) | 3,200 (1,600) | 9,072 (0) | 16 (40) | 45 (56) |
| 18 | M (F) | 400 (X) | 5,810 (X) | 9 (X) | 5 (X) |
| 19 | M (F) | 200 (1,600) | 0 (0) | 83 (12) | 83 (50) |
| 20 | M (F) | 0 (0) | 0 (0) | 68 (59) | 73 (74) |
| 21 | M (F) | 1,600 (3,200) | 31,825 (0) | 33 (83) | 55 (86) |
| 22 | M (F) | X (X) | 0 (x) | 39 (x) | 45 (x) |
| 23 | M (F) | 0 (6,400) | 0 (0) | 21 (63) | 30 (72) |
| 24 | M (F) | 3,200 (1,600) | 0 (0) | 42 (75) | 47 (71) |
| 25 | M (F) | 3,200 (6,400) | 1,546,984 (52,890) | 76 (51) | 75 (61) |
Statistical analysis
| Comparison | R2 |
|---|---|
| Malaria Ab titer versus HIV VL | 0.0002 |
| Malaria Ab titer versus % GIA (20% serum from HIV+) | 0.0008 |
| Malaria Ab titer versus % GIA (20% serum from HIV−) | 0.0284 |
| Malaria Ab titer versus GIA (10% serum from male) | 0.0004 |
| Malaria Ab titer versus GIA (10% serum from female) | 0.0337 |
Authors’ addresses: M. Kashamuka and N. Nzila, Project SIDA, PNLS/NRL/Hopital General de Kinshasa, Pavillion 27 BP 8502, Democratic Republic of the Congo and Division of Infectious Diseases, Johns Hopkins University School of Medicine. L. Mussey, Merck & Company, Inc., 795 Jolly Road, UN-C141, Blue Bell, PA, 19422. N. Lubaki, T. C. Quinn and R. Bollinger, Division of Infectious Diseases, Johns Hopkins University School of Medicine, Ross Research Building, 720 Rutland Avenue, Baltimore, MD 21205. N. Kumar, Department of Molecular Microbiology and Immunology, Johns Hopkins Bloomberg School of Public Health, 615 N. Wolfe Street, Baltimore, MD, 21205.
Acknowledgments: We thank all participants in this study and Cevayir Coban, Darin Kongkasuriyachai, Mobolaji Okulate, Mrinal Bhattacharyya, Sheila Keating, and Steven Renold for their help during these studies and Sabra Klein for help with statistical analysis. We also thank our colleagues from Project SIDA, especially Atibu Losoma, Milangu and Lelo for their contributions to collecting and processing specimens used in the study.
Financial support: This work was supported by grants to Johns Hopkins University from the Fogarty International Center of the U.S. National Institutes of Health (2-D 43 TW000010-AITRP) and the National Institutes of Allergy and Infectious Diseases, NIH (R03 AI 45119).
REFERENCES
- 3↑
Fowke KR, Kaul R, Rosenthal KL, Oyugi J, Kimani J, Rutherford WJ, Nagelkerke NJ, Ball TB, Bwayo JJ, Simonsen JN, Shearer GM, Plummer FA, 2000. HIV-1-specific cellular immune responses among HIV-1-resistant sex workers. Immunol Cell Biol 78 :586–595.
- 4↑
Hoffman SL, Sedegah M, Malik A, 1994. Cytotoxic T lymphocytes in humans exposed to Plasmodium falciparum by immunization or natural exposure. Curr Top Microbiol Immunol 189 :187–203.
- 5↑
Whitworth J, Morgan D, Quigley M, Smith A, Mayanja B, Eotu H, Omoding N, Okongo M, Malamba S, Ojwiya A, 2000. Effect of HIV-1 and increasing immunosuppression on malaria parasitemia and clinical episodes in adults in rural Uganda: a cohort study. Lancet 356 :1051–1056.
- 6↑
Kalyesubula I, Musoke-Mudido P, Marum L, Bagenda D, Aceng E, Ndugwa C, Olness K, 1997. Effects of malaria infection in HIV-1-infected Ugandan children. Ped Inf Dis J 16 :876–881.
- 7↑
Greenberg AE, Watso N, Ryder RW, Mvula M, Matadi N, Nsimba K, Matela B, Nsuami M, Davachi F, Hassig SE, 1991. Plasmodium falciparum and perinatally acquired human immunodeficiency virus type 1 infection in Kinshasa, Zaire. N Engl J Med 325 :105–109.
- 8↑
Nguyen-Dinh P, Greenberg AE, Mann JM, Kabote N, Francis H, Colebunders RL, Houng AY, Quinn TC, Davachi F, Lyamba B, Kalemba K, Embonga B, 1987. Absence of association between Plasmodium falciparum malaria and human immunodeficiency virus infection in children in Kinshasa, Zaire. Bull WHO 65 :607–613.
- 9↑
Allen S, Van de Perre P, Serufilira A, Lepage P, Carael M, Declercq A, Tice J, Black D, Nsengumuremyi F, Ziegler J, 1991. HIV and malaria in a representative sample of childbearing women in Kigali, Rwanda. J Inf Dis 164 :67–71.
- 10↑
Hoffman IF, Jere CS, Taylor TE, Munthali P, Dyer JR, Wirima JJ, Rogerson SJ, Kumwenda N, Eron JJ, Fiscus SA, Chakraborty H, Taha TE, Cohen MS, Molyneux ME, 1999. The effect of Plasmodium falciparum malaria on HIV-1 RNA blood plasma concentration. AIDS 13 :487–494.
- 11↑
Pisell TL, Hoffman IF, Jere CS, Ballard SB, Molyneux ME, Butera ST, Lawn SD, 2002. Immune activation and induction of HIV-1 replication within CD14 macrophages during acute Plasmodium falciparum malaria coinfection. AIDS 16: 1503–1509.
- 12↑
Kumar N, Folgar JP, Lubega P, 1992. Recognition of Plasmodium falciparum asexual stage antigens by antibodies in sera from people exposed to Plasmodium vivax. Am J Trop Med Hyg 47 :422–428.
- 14↑
Rhee MSM, Akanmori BD, Waterfall M, Riley EM, 2001. Changes in cytokine production associated with acquired immunity to Plasmodium falciparum malaria. Clin Exp Immunol 126 :503–510.
- 15↑
Dodoo K, Omer FM, Todd J, Akanmori BD, Koram KA, Riley EM, 2002. Absolute levels and ratios of proinflammatory and anti-inflammatory cytokine production in vitro predict clinical immunity to Plasmodium falciparum malaria. J Inf Dis 185 :971–979.