An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection

Summary

Human co-infection is difficult to replicate in vitro. However, human malaria parasites can readily be cultured in vitro, as can freshly isolated human peripheral blood mononuclear cells naturally infected with HIV. This provides an excellent model for studying early immune responses to malaria parasites in the context of HIV co-infection.

Abstract

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. Although understanding the disease dynamics for each of these pathogens independently is vital, it is also important that the interactions between these pathogens are investigated and understood.

We have developed a versatile in vitro model of HIV-malaria co-infection to study host immune responses to malaria in the context of HIV infection. Our model allows the study of secreted factors in cellular supernatants, cell surface and intracellular protein markers, as well as RNA expression levels. The experimental design and methods used limit variability and promote data reliability and reproducibility.

All pathogens used in this model are natural human pathogens (Plasmodium falciparum and HIV-1), and all infected cells are naturally infected and used fresh. We use human erythrocytes parasitized with P. falciparum and maintained in continuous in vitro culture. We obtain freshly isolated peripheral blood mononuclear cells from chronically HIV-infected volunteers. Every condition used has an appropriate control (P. falciparum parasitized vs. normal erythrocytes), and every HIV-infected donor has an HIV uninfected control, from which cells are harvested on the same day. This model provides a realistic environment to study the interactions between malaria parasites and human immune cells in the context of HIV infection.

Introduction

Co-infection, infection with multiple concurrent infections, is the norm in natural environments. Co-infection can have a major impact on disease pathology and on the clinical management of each infection. In the context of co-infection, vaccine and drug efficacy, as well as diagnostic testing, can be negatively impacted (reviewed in ¹). However, despite its importance, the majority of pathogen research considers only single infections.

Malaria and HIV-1 (HIV) are leading causes of morbidity and mortality globally. Areas of malaria and HIV endemicity share a wide geographic overlap, putting millions of people at risk of co-infect....

Protocol

This protocol requires the recruitment of donors for serum and RBC to be used for parasite culture, and HIV(+) and uninfected donors for PBMC isolation. Institutional Review Boards must approve all studies and all donors must provide informed consent prior to blood draw.

CAUTION: Working with human blood samples and human malaria parasites requires precautionary measures. Always wear a lab coat, gloves, and work in a level 2 Biosafety cabinet. In the event of accidental percutaneous exposure to human malaria, report to Health and Safety for prophylactic treatment. Additional safety consideration should also be put in place for working with ....

Results

The graphs depict the levels of IFNγ production from NKT cells (Figure 2), using CD56+CD3+γδ- gates to obtain the NKT cells population (data not shown). The cells were cultured for 72 hr prior to staining. Once stained, 100,000 CD3+ cells were acquired on the flow cytometer to obtain large enough populations of NK, NKT and γδ cells (cells of interest). A minimum of 5,600 NKT cells are displayed on each graph. TNF production is obtained in the same manner (data not shown). The graphs clearly demonstrate t.......

Discussion

Our protocol has been optimized in order to most realistically study HIV-malaria co-infection in vitro. First, fresh human RBCs and serum are required for malaria parasite culture. This is vital to obtain a healthy population of malaria parasites. Parasite lysates cannot be substituted for live parasites as cytokine production is much more rapid and intense when using live P. falciparum infected RBC (PfRBC) ^17,18. In addition, activation of cell types like NK cells, requires whole PfRBCs, and.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
alanine	Sigma	A7377
antibodies (see other table)
BD Cytofix/Cytoperm with BD GolgiPlug	BD	555028	includes brefeldin A, cytofix/cytoperm buffer and perm/wash buffer
BD Vacutainer ACD Solution A	BD	364506
BD Vacutainer Sodium Heparin	BD	17-1440-02
DPBS (no calcium, no magnesium)	Corning	21-031-CV
Fetal Bovine Serum	Sigma	F1051	heat inactivate before use
Ficoll-Paque PLUS	GE Healthcare	17-1440-02
gentamycin (10 mg/ml)	Gibco	15710-064
Hema3 Staining Set	Fisher	122-911
HEPES	Fisher	BP310-500
hypoxanthine	Sigma	H9636
Ionomycin	Sigma	I3909
MEM non-essential amino acids (10 mM)	Gibco	11140
PMA	Sigma	P8139
RPMI-1640 powder	Life-Technologies	31800-022
RPMI-1640 with L-glutamine and HEPES	Thermo Scientific	SH30255.01
sodium bicarbonate (powder, cell culture)	Sigma	S5761
Sodium Pyruvate (100 mM)	Gibco	11360
Tris (Trizma base)	Sigma	T6066
Trizol	Ambion	15596018
Trypan Blue (0.4%)	Gibco	15250-061
BD CompBead	BD	552843, 552845	depends on antibodies used
Parasite Gas Mixture	By special order		3% CO2, 1% O2, balance N2
Equipment
0.2 μm filter unit
Glass slides
T25 flasks
T75 flasks
50 ml tubes
24 well culture plates
unplugged Pasteur pipettes
plugged Pasteur pipettes
sterile transfer pipettes
hemocytometer
flow cytometer (3 laser)
Antibodies used for flow cytometry
Anti-TNF	eBiosciences	551487	FITC (fluorophore) 2 μl
Anti-IFNγ	Biolegend	506507	PE (fluorophore) 20 μl
Anti-CD8	Biolegend	300914	PE-Cy7 (fluorophore) 1 μl
Anti-CD14	Biolegend; BD Biosciences	325624; 551487	Biotin (fluorophore) 0.5 μl Streptavin PE-TR (fluorophore) 0.1 μl
Anti-CD56	Biolegend	318322	PerCP/Cy5.5 (fluorophore) 5 μl
Anti-γδ	Biolegend	331212	APC (fluorophore) 5 μl
Anti-CD3	Biolegend	300426	APC-Cy7 (fluorophore) 5 μl
Anti-CD4	Biolegend	317424	Pacific Blue (fluorophore) 1 μl