Plasmodium inui Infection Reduces the Efficacy of a Simian Immunodeficiency Virus DNA Vaccine in a Rhesus Macaque Model Through Alteration of the Vaccine-Induced Immune Response

Abstract

Human immunodeficiency virus type 1 and malaria are co-endemic in many areas. We evaluated the effects of Plasmodium inui infection on the performance of a simian immunodeficiency virus (SIV) DNA vaccine. Rhesus macaques were infected with P. inui by transfusion of whole blood from a persistently infected animal. Animals with and animals without P. inui infection were then vaccinated 4 times with an SIV DNA vaccine encoding SIVgag, SIVpol, and SIVenv. Animals were subsequently challenged with thirty 50% rhesus monkey infectious doses of SIVmac251 6 weeks after the last vaccination. P. inui–infected immunized animals showed a significantly higher viral load than animals without P. inui infection (P = .010, by the Wilcoxon rank sum test). The higher viral loads in the P. inui–infected animals were durable and were observed at all sampling time points across the study (P = .00245, by the Wilcoxon rank test). The P. inui–infected animals also had correspondingly lower CD4⁺ cell counts. There were fewer vaccine-specific CD4⁺ and CD8⁺ cells in the P. inui–infected animals, compared with uninfected animals. Of importance, P. inui infection seemed to decrease the number of CD8⁺ cells that could proliferate or secrete interferon γ, although the number of CD8⁺ cells capable of secreting tumor necrosis factor α following in vitro stimulation was increased. This study demonstrated that P. inui infection had an influence on the immune response to an SIV DNA vaccine and decreased the vaccine's efficacy.

Human immunodeficiency virus type 1 (HIV-1) is estimated to have infected >60 million individuals worldwide and has led to >20 million deaths. A highly effective vaccine is needed to stem this epidemic. More than 95 clinical trails of different HIV-1 vaccine candidates have been completed [1] with modest effects on infection [2]. Malaria, caused by infection with protozoan parasites of the genus Plasmodium, is also a significant global health problem and is responsible for the deaths of >1 million people annually, mainly in sub-Saharan Africa [3]. In fact, HIV-1 and malaria are coendemic in many regions, and there is concern that malaria will have an impact on an HIV-1 vaccine.

DNA vaccines have been demonstrated to be safe and immunogenic [4]. Our laboratory has completed a number of studies of HIV/simian immunodeficiency virus (SIV) DNA vaccines, using the macaque model [5–8]. We have developed an efficient DNA vaccine including SIVgag, SIVenv, and SIVpol genes that can suppress SIV replication after challenge [9]. By using this model, we wanted to determine the effect of ongoing Plasmodium inui infection on the efficacy of the DNA vaccine.

The impact of a low-level chronic Plasmodium infection on the performance of a DNA vaccine was assessed by leveraging a nonhuman primate model of P. inui infection and SIVmac251 challenge. The viral loads and CD4⁺ cell levels following SIVmac251 challenge were assessed. P. inui–infected animals exhibited higher viral loads and lower CD4⁺ cell numbers after challenge. We assessed the vaccine-specific immune response and found that the CD4⁺ and CD8⁺ cells capable of proliferating or secreting interferon γ (IFN-γ) in response to stimulation with vaccine antigen was significantly impacted. The altered vaccine-induced immune response was possibly due to the P. inui–infected animals having increased CD4⁺CD25⁺Foxp3⁺ regulatory T cells and an altered systemic cytokine profile. The data suggest that the altered systemic immune profile during P. inui infection can alter the vaccine response and decrease vaccine efficacy.

MATERIALS AND METHODS

Animals

Indian-origin rhesus macaques were housed at the Division of Veterinary Medicine at the Walter Reed Army Institute of Research (Silver Spring, MD) in accordance with the standards of the American Association for Accreditation of Laboratory Animal Care. Animals were allowed to acclimate for at least 30 days in quarantine prior to any experimentation. All animals received environmental enrichment throughout this study. All experimental procedures were performed under ketamine anesthesia, and all efforts were made to minimize pain and suffering. Immunological analysis was limited somewhat by blood volumes. We prioritized which assays would provide the most informative data at each time point. We then proceeded with what we considered the best analysis plan.

Infection and Immunization

Malaria Parasite Stock

P. inui OS strain (MR4-486) cryopreserved in glycerolyte in macaque red blood cells was obtained from the Malaria Research and Reference Reagent Resource Center (Manassas, VA). The sample was thawed, washed, and returned to isotonic media and then injected intravenously into 1 rhesus monkey. Seven days later, when parasitemia reached 2%, venous blood was taken, and multiple vials were cryopreserved in Glycerolyte 57 (Fenwal, Lake Zurich, IL) as a stock of malaria parasites for later use.

P. inui Infection of Experimental Monkeys

One donor monkey was infected with P. inui by intravenous injection of 1 vial of parasite stock. When parasitemia reached 1%, venous blood was taken from the donor and diluted in saline, and 1 × 10⁶ parasites were immediately injected intravenously into each experimental monkey.

Monitoring of Parasitemia

A total of 2 μL of blood was taken from the earlobe of each monkey by lancet stick every 2–3 days. This blood was spread as a monolayer on a glass slide and stained with Giemsa stain. Parasitemia was determined by examining 20 000 red blood cells at a final magnification of ×1000. Six P. inui–infected animals and 7 uninfected animals were vaccinated 4 times with 0.5 mg each of SIVgag, SIVpol, and SIVenv by electroporation on weeks 9, 13, 17, and 25.

DNA Vaccine and Peptides

The DNA vaccine used in this study expressed the modified rhesus macaque proteins for SIV gag (pSIVgag), pol (pSIVpol), or env (pSIVenv), as described elsewhere [7].

Regulatory T-Cell Assay

Fresh peripheral blood mononuclear cells (PBMCs) were isolated by standard Ficoll-Hypaque centrifugation. After cells were washed twice with phosphate-buffered saline (PBS), they were immunostained using a regulatory T-cell staining kit (eBioscience, San Diego, CA) according to the manufacturer's instructions. In brief, 10⁶ PBMCs were incubated with anti-CD3 APC-Cy7 (BD-Pharmingen, San Diego, CA), anti-CD4 APC, and anti-CD25 FITC for 30 minutes in the dark at 4°C. After permeabilization, cells were incubated with anti-Foxp3 PE at 4°C for at least 30 minutes in the dark. At some time points, permeabilized cells were incubated with anti-Foxp3 PE and anti-IFN-γ AF700 at the same time.

Enzyme-Linked Immunospot Assay (ELISpot) Assay for Interferon γ (IFN-γ)

ELISpot assays using IFN-γ reagents (MabTech, Sweden) and nitrocellulose plates (Millipore, Billerica, MA) were performed according to the manufacturer's instructions. A positive response was defined as detection of >50 spot-forming cells (SFCs)/10⁶ PBMCs. Each sample was performed in triplicate with peptides.

T-Cell Proliferation and Memory T-Cell Subset Assay

Fresh PBMCs were incubated with carboxyfluorescein succinimidyl ester (CFSE; 5 μmol/L) for 8 minutes at 37°C. Cells were then washed and incubated with medium alone (negative control), peptides at a concentration of 5 μg/mL, or concanavalin A (ConA; 5 μg/mL; positive control) for 5 days at 37°C in 96-well plates. Cultures without peptides were used to determine the background proliferative responses. PBMCs were immunostained with the following monoclonal antibodies: anti-CD3 APC-Cy7, anti-CD4 PerCP-Cy5.5, anti-CD8 APC, anti-CD95 PE-Cy5 (all BD-Pharmingen, San Diego, CA), and anti-CD28 ECD (Beckman Coulter, Fullerton, CA). Central and effector memory T cells were defined as CD28⁺CD95⁺ and CD28⁻CD95⁺ cells, respectively. Stained cells were washed in PBS and fixed (Cell-Fix). These cells were acquired on an LSRI instrument, using CellQuest software (BD Biosciences), and were analyzed with FlowJo software (Tree Star, Ashland, OR).

Cytokine Multiplex Assay

Plasma was analyzed for 28 cytokines and chemokines, using a cytokine monkey magnetic 28-plex panel kit (Invitrogen) according to the manufacturer's protocol. In brief, in a 96-well filter plate, 25 μL of antibody-coated beads, 50 μL of sample, and 50 μL of assay diluent or 100 μL of serially diluted cytokine standard were added to each well. Following a 2-hour period of incubation, the plate was washed and incubated with 100 μL of biotinylated detector antibody. The plate was washed twice, and 100 μL of streptavidin-RPE was added for 30 minutes. The assay plate was transferred to the Bio-Plex Luminex 100 XYP instrument for analysis.

Challenge and Determination of Viral Load and CD4⁺ and CD8⁺ T-Cell Levels

Animals were challenged 6 weeks after the last immunization with 30 monkey infectious doses (MID₅₀) of SIVmac251 by the intravenous route. Viral stocks were provided by Dr Nancy Miller (National Institute of Allergy and Infectious Diseases, National Institutes of Health) and titered at Bioqual (Rockville, MD). Plasma SIV RNA levels were determined by a quantitative RNA polymerase chain reaction [10]. Circulating CD3⁺, CD4⁺, and CD8⁺ T cells were quantitated by flow cytometry [10].

Intracellular Cytokine Staining

Fresh PBMCs were incubated with peptides for 5 hours at 37°C. A negative control (medium alone) and a positive control (Staphylococcus enterotoxin B [SEB], 1 µg/mL; Sigma-Aldrich) was included in each assay. Following incubation, the cells were washed and stained with surface antibodies: CD4 (PerCP-Cy5.5), CD3 (APC-Cy7), and CD8 (APC) (all BD Biosciences, San Jose, CA). Cells were then washed and fixed using the Cytofix/Cytoperm kit (BD Pharmingen, San Diego, CA), according to the manufacturer's instructions. Following fixation, the cells were stained with antibodies against the intracellular markers IFN-γ (Alexa Fluor-700) and tumor necrosis factor α (TNF-α; PE-Cy7) (all BD Biosciences, San Jose, CA). Cells were washed and fixed with PBS containing 1% paraformaldehyde. Stained and fixed cells were acquired on an LSRI instrument, using CellQuest software (BD Biosciences), and were analyzed with FlowJo software (Tree Star, Ashland, OR).

Statistic Analysis

For comparisons of IFN-γ ELISpots, T-cell proliferation, memory cells, intracellular cytokine staining, and regulatory T-cell level, 2-tailed Mann-Whitney U tests were performed using Prism Graphpad Software. For comparison of viral loads, the Wilcoxon rank test was performed. P values that were <.05 were considered indicative of a statistically significant difference.

RESULTS

Decreased Vaccine Efficacy Following SIV Challenge

To evaluate the effect of Plasmodium infection on vaccination, 6 macaques were initially infected intravenously with P. inui (infected group). Another 7 macaques were used as a control without P. inui infection (uninfected group). After 9 weeks, 2 groups of macaques were each immunized with DNA-based SIV vaccine 4 times, on weeks 9, 13, 17, and 25. All animals were challenged 6 weeks following the final immunization with 30 MIDs of SIVmac251 by the intravenous route. Subsequent to challenge, immunized animals coinfected with P. inui exhibited a viral load significantly higher than that of their uninfected counterparts (P = .010, by the Wilcoxon rank test). Higher viral loads in the P. inui–infected animals were durable and observed at all sampling time points across the study (P = .00245, by the Wilcoxon rank test) (Figure 1A). These results demonstrate that animals immunized with DNA vaccine induced a strong capacity to control viral replication and that P. inui infection decreased the capacity of the vaccine to control viral replication.

Figure 1.

Increased viral load after simian immunodeficiency virus (SIV) challenge in Plasmodium inui–infected macaques. All animals were challenged 6 weeks following the final immunization with 30 monkey infectious doses of SIVmac251. A, Plasma viral load after SIV challenge in macaques from the uninfected group (left) and the infected group (middle) and the average level in each group (right). B, CD4⁺ T cell amounts after SIV challenge in macaques from the uninfected group (left) and the infected group (middle) and the average level for each group (right).

After SIV challenge, we also tested the CD4 T-cell number [11–14]. The results showed that levels of CD4⁺ T cells in the P. inui–infected animals were significantly lower than in uninfected animals (P = .0143) (Figure 1B).

Decreased Cellular Immune Response in P. inui–Infected Macaques

To explore the effect of P. inui infection on SIV vaccine, we assessed the vaccine-induced immune response in all macaques by using an IFN-γ ELISpot assay. Macaques in the uninfected group showed an increasing IFN-γ response, from a mean of 1490 SFCs/10⁶ PBMCs after the first vaccination to a mean of 3527 SFCs/10⁶ PBMCs after the fourth vaccination (Figure 2A). In contrast, antigen-specific IFN-γ secretion after the fourth immunization reached 2856 SFCs/10⁶ PBMCs (Figure 2B) in the infected group. While there was a trend toward lower values in the infected animals, the difference in these numbers of antigen-specific IFN-γ responses to vaccine was not significant between groups (Figure 2C).

Figure 2.

Decreased simian immunodeficiency virus (SIV)–specific interferon γ (IFN-γ) responses in immunized Plasmodium inui–infected animals. IFN-γ–producing cells were assessed in immunized, uninfected animals (A) and in immunized P. inui–infected animals (B) after stimulation with SIV peptides (gag, env, or pol) for 24 hours, by enzyme-linked immunospot assay. C and D, Average level of IFN-γ–producing cells 2 weeks after the last immunization and (C) 2 weeks after challenge (D). PBMC, peripheral blood mononuclear cell; SFC, spot-forming cell.

In addition, assessment of antibody levels revealed no differences between groups at any time during the study (data not shown).

Suppressed Immune Response After SIV Challenge in Macaques Infected With P. inui

To further investigate why viral replication was higher in the macaques infected with P. inui, we assessed the immune response after challenge. SIV antigen–induced IFN-γ production and lymphocyte proliferation was assessed in PBMCs with or without antigen stimulation on weeks 2 and 20 after challenge. IFN-γ production in response to SIV antigen reached 3422 SFCs/million PBMCs and 2696 SFCs/million PBMCs in the uninfected group and the infected group, respectively, at week 2 after challenge (Figure 2D), which again was not a significant difference. Data from week 20 after challenge (data not shown) also did not demonstrate a significant difference. The data on IFN-γ production demonstrated that the IFN-γ response to SIV antigens did not show a significant difference before and after challenge in the 2 groups.

We next assessed lymphocyte proliferation. At 2 weeks after challenge, PBMCs were stimulated with SIV antigens for 5 days, resulting in a strong proliferative response by CD8⁺ T cells in the uninfected group. The response differed significantly between the uninfected and infected groups (mean response, 10.68% and 2% in the uninfected group and infected group, respectively) (P < .05) (Figure 3B). Compared with CD8⁺ T cells, the proliferative response of CD4+ T cells was low and reached a mean of 4.45% and 0.25% in the uninfected and infected groups, respectively (Figure 3B). Data on proliferation at week 20 after challenge did not show a significant change from that at week 2 (data not shown). These observations demonstrate that the number of CD8⁺ cells capable of proliferating in response to SIV antigen was significantly decreased in P. inui–infected macaques after SIV challenge.

Figure 3.

Suppressed proliferative capacity of simian immunodeficiency virus (SIV)–specific T cells in vaccinated Plasmodium inui–infected animals after the fourth immunization. Peripheral blood mononuclear cells (PBMCs) isolated from macaques 2 weeks after last immunization were stained with carboxyfluorescein succinimidyl ester and stimulated with growth medium, SIV antigens, or concanavalin (ConA) for 5 days. Following stimulation, cells were stained for phenotypic markers and analyzed by flow cytometry. A, Representative flow cytometry results showing proliferative capacity of CD4⁺ and CD8⁺ T cells from 1 macaque in each group. B, The mean proliferative capacity of CD4⁺ and CD8⁺ T cells is shown. *P < .05, by the unpaired t test.

Lower Frequency of Memory T Cells and IFN-γ Production Induced in Animals With P. inui Infection After Challenge

The frequency of proliferating memory T cell subsets in the 2 groups was compared using samples taken 20 weeks after challenge. Proliferating memory T cells were divided into 2 subsets by surface marker expression: central memory T cells (T_CM) were defined as CD28⁺CD95⁺ cells, and effector memory T cells (T_EM) were defined as CD28⁻CD95⁺ cells [15]. When the frequency of T_CM and T_EM in proliferating CD8⁺ cells (Figure 4B) was determined, an average of 13.78% and 8.9% T_CM were detected in proliferating CD8⁺ T cells isolated from animals in the uninfected and infected groups, respectively, and the percentages of T_CM did not differ significantly between the 2 groups (Figure 4B). However, a significantly higher percentage of T_EM in proliferating CD8⁺ T cells was observed in the uninfected group (average, 43.05%), compared with the infected group (average, 11.55%) (Figure 4B). In contrast, in proliferating CD4⁺ T cells, the percentages of T_CM and T_EM were not different between the 2 groups (data not shown). These observations suggest that P. inui infection results in a smaller percentage of T_EM among proliferating CD8⁺ T cells after SIV challenge.

Figure 4.

Induction of memory cell immune responses after challenge with SIVmac251. A, Strategy showing gating of total lymphocytes, live CD3⁺ cells, and CD4⁺ or CD8⁺ memory T cells. B, Expression of central memory T cells (T_CM) and effector memory T cells (T_EM), defined as CD28⁺CD95⁺ and CD28^–CD95⁺, respectively, in proliferating CD8⁺ lymphocytes on week 20 after challenge.

To better determine the quality of the CD8⁺ cell immune response, SIV-specific CD8⁺ cells were assessed by flow cytometry for the production of IFN-γ and TNF-α. PBMCs were isolated from rhesus macaques 20 weeks after challenge and stimulated for 5 hours with growth medium alone, SIV peptides, or SEB. Polyfunctional cytokine analysis showed IFN-γ production among more CD8⁺ T cells from animals without P. inui infection than among cells from the P. inui–infected group (Figure 5B). In contrast, the percentage of CD8⁺ T cells producing TNF-α was higher in P. inui–infected macaques (mean, 2.07%) than in the uninfected group (average, 0.05%) (Figure 5C). Production of these cytokines in CD4⁺ T cells did not differ significantly between the 2 groups (data not shown). Taken together, the production of IFN-γ was decreased in macaques with P. inui infection, whereas production of TNF-α was increased in these macaques.

Figure 5.

Tumor necrosis factor α (TNF-α) is upregulated in Plasmodium inui–infected macaques. A, Strategy showing gating of total lymphocytes, live CD3⁺ cells, and CD4⁺ or CD8⁺ T cells and expression of the cytokines interferon γ (IFN-γ; B) and tumor necrosis factor α (TNF-α; C) in CD8⁺ lymphocytes on week 20 after challenge. Data are presented as mean values ± SD. *P < .05, by the unpaired t test.

Increased Regulatory T Cells in P. inui–Infected Macaques

The level of regulatory T cells was determined in all macaques at week 9 after P. inui infection and prior to vaccination. The average percentage of regulatory T cells in the infected group and uninfected group was 1.86% and 1.34%, respectively (Figure 6A). After the fourth vaccination with a DNA-based SIV vaccine, P. inui–infected animals were again observed to have a higher percentage of regulatory T cells than their uninfected counterparts (average, 2.2% and 1.4% in the infected group and uninfected group, respectively) (Figure 6A). Data from both time points revealed significant differences between the infected group and uninfected group. Taken together, the results indicate that P. inui infection increased the frequency of regulatory T cells in macaques. To determine whether the expression of regulatory T cells was affected by the challenge, we examined the percentage of regulatory T cells 5 months after SIV challenge and found that the frequency of regulatory T cells did not show a significant change after SIV challenge. After challenge, the regulatory T cell level in P. inui–infected animals was significantly higher than that in uninfected animals (P = .040) (Figure 6A).

Figure 6.

High percentage of regulatory T cells in Plasmodium inui–infected immunized animals after challenge with SIVmac251. A, Percentage of CD25⁺Foxp3⁺ regulatory T cells in a CD4⁺ T cell gate in immunized P. inui–infected and -uninfected macaques after challenge. B, Effector phenotype of Foxp3⁺ regulatory T cells in a CD4⁺ T cell gate in immunized P. inui–infected and -uninfected animals after challenge. Data are presented as mean values ± SD. Abbreviation: IFN-γ, interferon γ. *P < .05, by the unpaired t test.

In addition, we analyzed the phenotype of regulatory T cells by costaining of IFN-γ and Foxp3. Most regulatory T cells did not show the IFN-γ effector phenotype. Furthermore, the IFN-γ⁻ regulatory T cells were highly expressed in P. inui–infected macaques (Figure 6B). Only 0.16% and 0.3% of CD4⁺ cells in infected and uninfected macaques, respectively, expressed the phenotype of Foxp3⁺IFN-γ⁺ (Figure 6B). In Foxp3⁻CD4⁺ T cells, uninfected macaques induced high IFN-γ secretion (Figure 6B). Together, these data suggest that, during SIV infection, Foxp3⁺ regulatory T cells were still highly expressed in P. inui–infected macaques, and fewer regulatory T cells acquired the capacity to express IFN-γ.

Altered Cytokine and Chemokine Milieu in P. inui–Infected Macaques

The expression of 28 cytokines and chemokines in the plasma of all macaques was evaluated at week 9 after P. inui infection and prior to vaccination. There was a significant difference in the cytokine/chemokine profile between the groups, with 5 of the 28 proteins measured (Figure 7) showing a difference. Results indicated that the type 2 helper T-cell cytokine interleukin 4 (IL-4) was more highly expressed in infected macaques, compared with uninfected macaques. IL-4 can have a significant impact on the induction of IL-12 and IFN-γ, downmodulating the type 1 helper T-cell response and potentially the cellular immune response.

Figure 7.

Expression of type 2 helper T cell (Th2)–related cytokines and chemokines in Plasmodium inui–infected macaques. Plasma was taken from macaques 9 weeks after P. inui infection and prior to vaccination and was analyzed for 28 cytokines and chemokines, using a cytokine monkey magnetic 28-plex panel kit (Invitrogen) according to the manufacturer's protocol. Data are levels of the Th2-related cytokines interleukin 4 (IL-4), MDC, interleukin 1b (IL-1b), RANTES, MIG, and MCP-1 and the type 1 helper T cell (Th1)–related cytokines interleukin 12 (IL-12), interleukin 15 (IL-15), interleukin 2 (IL-2). Each macaque is indicated by a dot, and the average of every group is indicated by a short line.

The molecules that showed the greatest difference involved cytokines and chemokines associated with trafficking and the inflammatory response. Interleukin 1β, a member of the interleukin 1 cytokine family, is produced by activated macrophages and is an important mediator of the inflammatory response. RANTES, the molecule responsible for recruiting leukocytes into inflammatory sites, was also more highly induced and reached a level of 2850 pg/mL in infected macaques, compared with 1140 pg/mL in uninfected macaques (Figure 7). MIG (also known as CXCL9) and MDC (also known as CCL22) were both upregulated in macaques infected with P. inui (Figure 7).

DISCUSSION

P. inui infection suppressed vaccine efficacy. Infection of rhesus macaques with P. inui resulted in an increased population of CD4⁺CD25⁺Foxp3⁺ regulatory T cells and altered the cytokine and chemokine milieu. Regulatory T cells can suppress the antiviral immune responses, such as the IFN-γ response [16, 17]. Additionally, alterations in cytokine levels can clearly impact the immune responses. While we observed a slight decrease in the number of IFN-γ–producing cells when total PBMCs were assessed, analysis of CD8⁺ cells in more detail by flow cytometry revealed significant differences. In addition, an efficacious vaccine-associated cellular immune response is dependent on the ability of CD8⁺ cells to proliferate rapidly following infection. In a previous study, we found that interleukin 15 could enhance proliferation and was associated with viral suppression [18]. Results from the current study demonstrated that establishment of P. inui infection decreased the antigen-specific IFN-γ response as well as the T cell proliferative responses induced by an SIV DNA-based vaccine (Figures 1–3).

Many studies of malaria and HIV-1 disease indicate that malaria is more common and severe in HIV-1-infected adults in Africa [19–21], suggesting that HIV-1 infection increases susceptibility to malaria. Cuadros et al demonstrated that malaria might be a risk factor for concurrent HIV-1 infection [22]. There are observations that acute malaria elevated the HIV-1 load by as much as 1 log in some subgroups [23, 24]. Other studies suggest that malaria is less strongly associated than other opportunistic infections with HIV-1-associated immunosuppression [25].

Our observations suggest that regulatory T cells induced by P. inui infection may in part be responsible for the suppressed vaccine-induced immune responses and increased viral load after SIV challenge. Although the functional mechanisms used by regulatory T cells are complex and still incompletely understood, there is increasing evidence that regulatory T cells use different mechanisms to regulate immune responses in lymphoid and nonlymphoid tissues. This concept is best exemplified by the distinct phenotypes in mice that selectively lack regulatory T cell expression of either interleukin 10 (IL-10) or cytotoxic T lymphocyte antigen 4 (CTLA4). IL-10 is a cytokine that is produced by regulatory T cells and can both directly and indirectly inhibit effector T cell responses during infection, autoimmunity, and cancer [26–28]. Deletion of IL-10 selectively in regulatory T cells resulted in the development of colitis and exaggerated immune responses at other environmental interfaces, such as the skin and lungs [29]. By contrast, loss of CTLA4 expression in regulatory T cells resulted in severe lymphoproliferative disease characterized by massive lymphadenopathy and splenomegaly and was associated with the accumulation of CD4⁺CD44^hiFoxp3^– effector T cells, spontaneous multiorgan autoimmunity, and early death [30].

As noted above, cytokines and chemokines associated with trafficking and the inflammatory response were upregulated by P. inui infection. The potential consequence of the higher concentrations of chemokines could have lead to the upregulation of TNF-α production. TNF-α is a pleiotropic cytokine that acts as an immune and inflammatory mediator. However, it has been suggested that TNF-α is involved in the pathogenesis of HIV-1 infection since it is overproduced by infected individuals [31–33]. Therefore, the switch in the immunological profile from IFN-γ to TNF-α may have had deleterious effects.

In conclusion, infection with P. inui results in compromise of the immune response in animals vaccinated with a SIV DNA vaccine. The data suggest that a possible mechanism for the decremented response to the vaccine involves erosion of the cellular response in animals with malaria. These observations may have implications for the design and evaluation of HIV-1 vaccines in areas where malaria is endemic.