SIV infection aggravates malaria in a Chinese rhesus monkey coinfection model
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The co-occurrence of human immunodeficiency virus (HIV) infection and malaria in humans in endemic areas raises the question of whether one of these infections affects the course of the other. Although epidemiological studies have shown the impact of HIV infection on malaria, the mechanism(s) are not yet fully understood. Using a Chinese rhesus macaque coinfection model with simian immunodeficiency virus (SIV) and Plasmodium cynomolgi (Pc) malaria, we investigated the effect of concurrent SIV infection on the course of malaria and the underlying immunological mechanism(s).
We randomly assigned ten Chinese rhesus monkeys to two groups based on body weight and age. The SIV-Pc coinfection animals (S + P group) were infected intravenously with SIVmac251 eight weeks prior to malaria infection, and the control animals (P group) were infected intravenously with only Pc-infected red blood cells. After malaria was cured with chloroquine phosphate, we also initiated a secondary malaria infection that lasted 4 weeks.
We monitored body weight, body temperature and parasitemia, measured SIV viral loads, hemoglobin and neopterin, and tracked the CD4+, CD8+, and CD4+ memory subpopulations, Ki67 and apoptosis by flow cytometry. Then, we compared these parameters between the two groups.
The animals infected with SIV prior to Pc infection exhibited more severe malaria symptoms characterized by longer episodes, higher parasitemia, more severe anemia, greater body weight loss and higher body temperature than the animals infected with Pc alone. Concurrent SIV infection also impaired immune protection against the secondary Pc challenge infection. The coinfected animals showed a reduced B cell response to Pc malaria and produced lower levels of Pc-specific antibodies. In addition, compared to the animals subjected to Pc infection alone, the animals coinfected with SIV and Pc had suppressed total CD4+ T cells, CD4+CD28highCD95high central memory T cells, and CD4+CD28lowCD95− naïve T cells, which may result from the imbalanced immune activation and faster CD4+ T cell turnover in coinfected animals.
SIV infection aggravates malaria physiologically and immunologically in Chinese rhesus monkeys. This nonhuman primate SIV and Pc malaria coinfection model might be a useful tool for investigating human HIV and malaria coinfection and developing effective therapeutics.
KeywordsPlasmodium cynomolgi Simian immunodeficiency virus (SIV) Coinfection Rhesus macaques Antibody Cellular immune response Neopterin Immune activation Turnover
acquired immunodeficiency syndrome
human immunodeficiency virus
infected red blood cells
simian immunodeficiency virus
median tissue culture infectious dose
central memory T cells
effector memory T cells
naïve T cells
World Health Organization
Malaria is a life-threatening infectious disease that is prevalent in sub-Saharan Africa and caused by Plasmodium parasites. The most recent statistics show that there were 216 million malaria cases and 0.5 million recorded malaria deaths worldwide in 2016, approximately 90% of which occurred in Africa . Human immunodeficiency virus (HIV) targets and impairs immune defenses against infections and some type of cancers, leading to acquired immunodeficiency syndrome (AIDS). WHO data indicate that approximately 70% of the 36.7 million HIV patients worldwide live in Africa . Given that the endemic regions of Plasmodium and HIV infection overlap extensively and that many people are infected, there is an increased risk of coinfection with these two pathogens.
Because these two infections have a profound impact on human health, many studies have been conducted to investigate the potential interactions between them. Although some early studies failed to observe any direct associations between HIV and malaria [3, 4], several recent studies have shown that concomitant HIV infection worsens malaria. The incidence of malaria infection is increased in HIV-endemic regions and in pregnant women [5, 6], and more severe clinical symptoms of malaria occur in HIV+ adults with partial immunity to the parasites [7, 8]. Limited clinical and experimental studies showed that impaired immune activation, reduced anti-malaria antibody production and subsequent immunosuppression were associated with an increased frequency of clinical malaria and parasitemia in HIV-infected individuals [9, 10, 11, 12, 13, 14]. However, the immunological interaction between these two important infectious diseases is not fully understood.
Previous studies on the interaction between HIV infection and malaria in humans showed disparate results and often reached inconsistent conclusions, probably due to the confounding factors involved in human studies. Nonhuman primates have been used as a suitable model for the study of human malaria HIV infection. Rhesus macaques can be infected by several Plasmodium species and by simian immunodeficiency virus (SIV) [15, 16]. Plasmodium cynomolgi (Pc) infection in monkeys is an established model for the study of Plasmodium vivax infections in humans [17, 18]. SIV infection in Chinese rhesus macaques shows many features that are similar to those of HIV infection in humans . We established a Chinese rhesus macaque coinfection model to study how Pc malaria modulates the course of SIV infection under controlled experimental conditions . Using a similar coinfection model, we investigated the modulatory effects of SIV infection on the course of Pc malaria. We observed that pre-existing SIV infection profoundly modulates the course and severity of primary and secondary blood-stage Pc malaria. In addition, SIV infection alters the humoral immune response, the homeostasis of CD4+ T cells and immune activation during the acute phase of Pc malaria, which may lead to impaired immunity and exacerbated malaria.
Adult Chinese rhesus macaques (Macaca mulatta) used in this study, 5–6 years of age, were purchased from a commercial breeding farm (Jiufo Monkey Farm) in Guangzhou, China. All animals were confirmed to be free of Plasmodium, B virus, D-type simian retrovirus, simian T-lymphotropic virus type 1 and SIV. The animals were housed at the Non-Human Primate Animal Center of the Guangzhou Institutes of Biomedicine and Health (GIBH). The animal experiments were designed and performed in accordance with the NIH Guide for the Care and Use of Laboratory Animals, and the protocols were approved by the GIBH Institutional Animal Care and Use Committee. Each animal was housed in a separate cage, received standard primate feed and fresh fruit or eggs daily, and had free access to water. The animals were monitored routinely for body temperature, body weight, physical fitness and blood biochemistry. After this experiment, the macaques were included into another coinfection study. They were euthanized when they presented with advanced stages of AIDS; criteria for euthanasia included 15% weight loss in 2 weeks, unresponsive opportunistic infection, persistent anorexia, intractable diarrhea, progressive neurologic signs or other serious illness. Macaques were euthanized via infusion of sodium pentobarbital after anesthesia with ketamine HCl by a veterinarian.
Pc malaria and SIV infection procedures
Parasitemia was monitored daily by preparation and microscopic examination of blood films stained with Giemsa stain (Sigma-Aldrich, St. Louis, Missouri, USA). Body temperature was measured in the inner ear twice daily with a medical digital ear thermometer.
Blood collection and preparation
Blood for parasitemia was obtained by pricking a finger or earlobe. Both thick and thin blood films were prepared . Venous blood was incubated at room temperature for 2 h to allow clotting, and serum was carefully aspirated. K2-EDTA anticoagulated venous blood was centrifuged at 1000×g, and the supernatant (plasma) was collected for SIV RNA isolation. K2-EDTA anticoagulated venous whole blood was used for flow cytometry.
Plasma viral RNA levels were determined using a SYBR Green-based real-time PCR protocol, as previously described . The viral RNA was purified with a QIAamp Viral RNA Mini Kit (Qiagen, Valencia, CA, USA), and real-time PCR was carried out with a one-step QuantiTect SYBR Green RT-PCR Kit (Qiagen, Valencia, CA, USA) on CFX96 Real-Time PCR Detection System (Bio-Rad, Hercules, California, USA).
B cells and T cell subpopulations in peripheral blood were analyzed by flow cytometry. The following fluorescence-conjugated antibodies were used: anti-CD3-PerCP, anti-CD4-FITC, anti-CD4-PE-Cy7, anti-CD4-PerCP (L200), anti-CD8-APC-Cy7 (RPA-T8), anti-CD45-PE (DO58–1283), anti-CD28-FITC (CD28.2), anti-CD95-APC, anti-CD95-PE Cy5 (DX2) and anti-Ki-67-PE (BD Pharmingen, San Diego, CA, USA), anti-CD8-APC and anti-CD8-PE (B9.1) (Immunotech SAS, Marseille Cedex, France). Cell surface marker staining was performed as follows: 50 μl of EDTA-treated whole blood was stained with antibodies at room temperature (RT) for 20 min in the dark, and red blood cells were then lysed in lysis buffer (BD Pharmingen, San Diego, CA, USA) at RT for 10 min, followed by a wash with PBS containing 2% FBS. The number of lymphocytes in peripheral blood samples was determined using the BD TruCOUNT Kit (BD Biosciences, San Jose, CA, USA) according to the manufacturer’s instructions. For intracellular Ki-67 staining, the cells were fixed and permeabilized using the BD Fixation/Permeabilization Solution Kit according to the manufacturer’s instructions, with surface marker staining as described above. Apoptosis and cell death were detected with a PE annexin V apoptosis detection kit (BD Pharmingen, San Diego, CA, USA) according to the manufacturer’s instructions. The stained cells in all assays were stored in the dark at 4 °C and immediately analyzed by flow cytometry on either a FACSCalibur or FACSAria (BD Biosciences, San Jose, CA, USA) system, and the measurements of all samples were completed within 4 h after the staining. The data were analyzed using FlowJo software (Tree Star. Inc., USA).
Serum levels of Pc-specific antibodies were determined by ELISA. ELISA plates (Nunc A/S, Denmark) were coated with soluble Pc antigens prepared as described previously  at a concentration of 6 μg/ml in PBS overnight at 4 °C. The plates were blocked with 1% bovine serum albumin in PBS for 1 h. Individual serum samples were subjected to twofold serial dilution, and 50 μl of each dilution was added to the plate and incubated for 2 h at RT. After extensive washing, a horseradish peroxidase-conjugated goat anti-rhesus IgG (H + L) antibody (SouthernBiotech, Birmingham, AL, USA) was added and incubated at RT for 2 h. Reactivity was visualized with the 3′,3′,5′,5′-tetramethylbenzidine substrate and stopped with 100 μl of a 1 M H2SO4 solution. OD values were read at 450 nm microplate reader (Bio-TEK, Winooski, Vermont, USA).
We determined the neopterin concentration in plasma collected 0, 1, 2 and 5 weeks after Pc inoculation with an ELISA kit (Alpha Diagnostic, San Antonio, Texas, USA) according to the instructions provided in the manual.
GraphPad Prism (Version 6) was used for statistical analysis. Intergroup comparisons were performed using an unpaired t test. Pearson’s correlation analysis was performed to analyze the data for viral load, antibody levels and neopterin levels. Analysis of body weight changes before and after Pc inoculation was performed using multiple comparisons with the Sidak correction after repeated measures two-way ANOVA, and the adjusted P value was reported. Data are reported as the mean ± SD. P values less than 0.05 were considered statistically significant.
Modulation of malaria by concurrent SIV infection
All animals were treated with chloroquine at week 26 of Pc infection to terminate malaria and were reinfected with Pc parasites 4 weeks later (Fig. 1). The S + P group animals developed malaria episodes, but all the animals in the P group controlled the parasites to a very low level (parasites could be observed only in thick smear slides) (Fig. 2b, c, Additional file 1: Figure S1).
Alteration of the humoral immune response to malaria
Responses of the T cell subset
Further analysis of the response of CD4+ subpopulations (Additional file 4: Figure S4) showed that the changes in the number of central memory T cells (TCMs, defined as CD4+CD28highCD95high) showed a pattern similar to those of the total CD4+ T cells. The animals in the S + P group had significantly weaker CD4+ TCM responses than the animals in the P group over most of the duration of the coinfection (Fig. 4d). CD4+ effector memory T cells (TEMs, defined as CD4+CD28-CD95+) responded to Pc infection in the two groups of animals in a similar manner, and no significant difference was observed between the two groups (Fig. 4d). CD4+ naïve T cells (TNs, defined as CD4+CD28lowCD95−) increased following Pc infection in the P group animals, but they decreased after SIV infection in S + P animals and did not increase after Pc infection. The TN cell count in the P group animals was significantly higher than that in the S + P group animals from weeks 11 to 36 of Pc infection (Fig. 4d).
Immune activation and CD4+ T cell dynamics during the acute phase of pc infection
We established a nonhuman primate SIV and Pc malaria coinfection model to investigate the influence of concurrent SIV infection on the pathogenesis of malaria and the associated immunological responses under defined experimental conditions. We observed that compared with the animals subjected to Pc infection alone, Chinese rhesus monkeys with pre-existing SIV infection exhibited aggravated malaria symptoms following infection with the Pc malaria parasite. The S + P coinfected animals experienced longer episodes of malaria with higher peak levels of parasitemia, developed more severe anemia, and experienced greater weight loss and a slower recovery. In addition, the coinfected animals developed impaired CD4+ T cell responses and suppressed B cell proliferation and antibody production in response to primary Pc infection. Furthermore, the activation and apoptosis of CD4+ T cells were higher in S + P coinfected animals than in animals subjected to Pc infection alone. These impaired humoral and cellular immune responses resulting from the imbalanced immune activation in coinfected animals may contribute to the reduction of protective immunity to a level that is unable to efficiently control primary or secondary Pc malaria infection.
Animals infected with SIV or Pc in our model developed symptoms and disease progression that were similar to those of HIV infection or malaria in humans. The animals coinfected with Pc during chronic SIV infection experienced rapid parasite growth, which we had to control with several low doses of artesunate treatment to prevent animal from death. This low dose of artesunate cannot eliminate the parasites. After the treatment(s), parasites continued to replicate for several weeks in both groups and finally suppressed by host immunity. These observations indicated that the low doses of artesunate treatment in this study did not affect the results fundamentally. Furthermore, despite the drug treatment, the coinfected animals experienced more severe anemia and greater weight loss and took a longer time to recover from anemia and weight loss than the animals subjected to infection with malaria alone. These characteristics were very close to those of P. fragile-SHIV coinfected animals  or patients with HIV and malaria coinfection [5, 25, 26]. Our results were consistent with observations in humans that HIV-positive patients lacking immunity to malaria were more likely to suffer from severe malaria than HIV-negative individuals.
In addition to investigating the impact of SIV infection on primary Pc malaria, we further analyzed the effect of concurrent SIV infection on secondary Pc malaria. After clearing the primary malaria with chloroquine, the animals were reinfected with Pc parasites to initiate secondary malaria. We found that the animals in the P group exhibited immune protection and control of secondary Pc malaria, which was similar to the naturally acquired partial immunity reported in human adults in malaria endemic areas . However, the animals in the S + P group developed mild clinical episodes with high levels of parasitemia. These results demonstrate that SIV infection acts as a risk factor for severe malaria. It has been reported that human patients who are naïve to malaria and infected with HIV are more likely to display severe malaria than HIV-negative persons. However, in individuals who are semi-immune to malaria, no significant difference in the risk of severe malaria was found between the HIV-infected and HIV-negative individuals . Our observations show that SIV-positive animals developed more severe malaria symptoms during both primary and secondary Pc infection. This inconsistency is likely due to variations in the interval between the two infections and differences in the immune status of clinical patients.
The humoral antibody response is an important part of the immune effector mechanism against malaria. Studies in humans and monkey models demonstrated that concomitant HIV/SIV infection impairs anti-malarial antibody production [10, 11, 14]. Our results showed that Pc malaria infection induced robust proliferation of B cells and a strong antibody response in animals in the P group. These humoral immune responses were suppressed in S + P coinfected animals. HIV infection can directly impair B cell responses by exhausting memory B cells . Furthermore, parasite infection can exacerbate this expansion of atypical memory B cells in patients coinfected with HIV and malaria . Our results demonstrated that the S + P animals with a higher SIV viral load on the day of Pc inoculation produced lower levels of anti-Pc antibodies. Pre-existing SIV infection during primary Pc infection may impair antibody production, resulting in less effective immune protection for the control of both primary and secondary Pc malaria.
CD4+ T cells are essential for cytokine production, B cell activation and antibody production and phagocyte activation. Our results showed that the CD4+ T cell response to Pc malaria was significantly impaired in the S + P group animals, but the CD8+ T cell response was not affected. As observed by others , in our study, the numbers of TCMs and TNs decreased after SIV infection and showed a weaker response to Pc infection in the S + P group animals. A continuous decline in TCMs and TNs may result in delayed replenishment of exhausted TCMs or TEMs [29, 30]. Short-lived CD4+ TEM cells are believed to play a key protective role in controlling parasitemia [30, 31, 32]. We noticed in our study that the change in CD4+ TEM numbers was inversely associated with parasite density in both groups. Although this difference was not statistically significant, a higher number of CD4+ TEMs was detected in the P group animals than in the S + P animals. These TEMs may contribute to immune protection against Pc parasites during primary and secondary infection. We propose that the rapid exhaustion of CD4+ TEMs and the lack of replenishment of TEMs from the TN or TCM pool may lead to compromised immune protection against Pc malaria in S + P coinfected animals.
In this study, we evaluated Th1 immune activation by measuring the level of neopterin in the plasma during the acute stage of Pc malaria infection. We found that the levels of neopterin in both groups of animals increased following Pc inoculation. The higher level of neopterin in S + P animals on the day of Pc inoculation indicated that the SIV-infected animals were in a state of immune activation. The S + P animals maintained higher levels of neopterin during the acute phase of Pc infection. Chronic immune activation in HIV infection is one reason for the persistent decline of CD4+ T cells due to senescence and apoptosis, and eventually death, of these cells. We further analyzed the data on CD4+ T cell dynamics, including activation, proliferation and apoptosis. The frequencies of Ki-67+ among CD4+ T cells, TCMs, TEMs and TNs and the frequency of annexin V+ CD4+ T cells were higher in the S + P group than in the P group, even though only the difference in the frequency of Ki-67+CD4+ TCMs was statistically significant (Fig. 5). Unlike mouse studies, which can be easily repeated or which can increase the number of animals to examine whether the absence of statistical significance is due to an insufficient number of animals, we could not easily repeat our monkey model study or increase the number of animals. Therefore, we further analyzed the correlations among the above parameters related to CD4+ T cell dynamics based on significant differences in neopterin levels and Ki-67+CD4+ TCM frequency between the two groups. The analysis indicated that these parameters were correlated with each other (Fig. 6), suggesting that a rapid turnover of CD4+ T cells also existed in our S + P group animals. Therefore, SIV infection in S + P animals may result in aberrant immune activation and disease progression, as reported in HIV+ humans and SIV-infected macaques or sooty mangabeys [33, 34, 35, 36, 37], and eventually lead to an impaired immune response to Pc malaria.
Overall, the present study demonstrated that SIV infection acted as a risk factor for primary and secondary Plasmodium infection. Severe malaria in coinfected animals might result from impaired humoral immunity and CD4+ T cell responses against parasites. These observations suggest that appropriate HIV therapy may be needed not only to alleviate clinical malaria in HIV-positive patients but also to reduce the risk of plasmodium parasite spreading in areas with a moderate or high prevalence of HIV and malaria.
We are grateful to the staff at the Laboratory Animal Center and Instrument Center for collecting blood samples, preparing blood smear slides and monitoring the body temperature and body weight of the animals. We thank Caijun Sun, Yaozeng Lu, Chuan Qin, and Zhiwei Chen for valuable help with the study.
GL performed the experiments, analyzed the data and wrote the manuscript. YL performed the experiments, analyzed the data and drafted the manuscript. LQ designed the study and performed the experiments. YYan performed the FACS experiments and analyzed the data. YYe and YC performed ELISA and parasite-related experiments. CH and SZ processed the blood samples. YYao contributed to performing the experiments. ZS and XC conceived the experiments and critically reviewed and revised the manuscript. All authors read and approved the final manuscript.
This work was supported by grants from the Ministry of Sciences and Technology Key Program (2016YFE0107300), the Ministry of Science and Technology of the People’s Republic of China (2006CB504200), the National Natural Science Foundation of China (81673003, 31570925) and Guangzhou Municipal Science and Technology Project (201707010447) to XC and a grant from the Chinese Academy of Sciences (KSCX2-YW-R-164) to ZS. YCY is sponsored by the Shenzhen Science and Technology Program (JCYJ20150401171904130) and the Postdoctoral Science Foundation (2016 M592571). None of the funding bodies were involved in the design of the study, data collection, analysis, interpretation of data, and in writing the manuscript.
Ethics approval and consent to participate
The study was reviewed and approved by the Institutional Animal Care and Use Committee at Guangzhou Institutes of Biomedicine and Health (GIBH), the Chinese Academy of Sciences, in accordance with the NIH Guide for the Care and Use of Laboratory Animals.
Consent for publication
The authors declare that they have no competing interests.
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