Name: an in vitro model for measuring immune responses to malaria in the context of hiv co-infection
Uploaded: 2015-10-06T14:00:00
Description: Watch this Scientific Journal Video about An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection at JoVE.com

C.A.M.F. and L.S. participated in protocol design, acquisition and analysis of data, and drafting of the article. 
The authors wish to thank Dr. Kain, Dr. Loutfy, Dr. Wasmuth, and Dr. Ayi for their contributions.
C.F. was supported by a CTN/Ontario HIV Treatment Network (OHTN) postdoctoral fellowship. L.S. is supported by an OHTN Junior Investigator Development Award. The present work was supported by a Canadian Institutes of Health Research (CIHR) operating grant (MOP-13721 and 115160), and a CIHR New Investigator Catalyst grant.

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 t...

<ol>
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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...

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-infection and consequently at risk for more severe clinical disease 2&#8211;10. The two diseases negatively interact. In HIV-infected individuals, higher HIV viral loads and temporary decreases in CD4+ T-cell counts can be seen during a malaria infection, while malaria parasite burdens and risk of clinical and severe malaria are higher in co-infected individuals 2,3,5,7,8,10. The mechanisms by which HIV increases malaria severity are not fully understood and warrant further investigation.
Here we describe a method by which malaria and HIV co-infection can be studied in vitro. Specifically, this method allows for the examination of malaria-specific immune responses in the context of HIV infection. Our protocol describes a versatile co-culture system using freshly isolated peripheral blood mononuclear cells (PBMCs) isolated from chronically HIV infected donors and in vitro cultured P. falciparum parasitized erythrocytes (PfRBC). The impact of HIV antiretroviral therapy on these responses can also be examined using prospectively collected PBMCs from HIV(+) donors pre- and post-therapy.
We have used this system to investigate the impact of HIV infection on malaria-specific innate immune responses 11,12, and were able to determine that malaria-specific IFN&#947; and TNF responses are impaired in NK cells, NKT cells, &#947;&#948; T cells from HIV(+) donors pre- and post-HIV antiretroviral therapy. Additionally, we were able to use this system to determine that monocytic functions are also impaired in HIV(+) donors, but recover post-HIV antiretroviral therapy.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>alanine</td>
			<td>Sigma</td>
			<td>A7377</td>
			<td></td>
		</tr>
		<tr>
			<td>antibodies (see other table)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>BD Cytofix/Cytoperm with BD GolgiPlug</td>
			<td>BD</td>
			<td>555028</td>
			<td>includes brefeldin A, cytofix/cytoperm buffer and perm/wash buffer</td>
		</tr>
		<tr>
			<td>BD Vacutainer ACD Solution A</td>
			<td>BD</td>
			<td>364506</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Vacutainer Sodium Heparin</td>
			<td>BD</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>DPBS (no calcium, no magnesium)</td>
			<td>Corning</td>
			<td>21-031-CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum</td>
			<td>Sigma</td>
			<td>F1051</td>
			<td>heat inactivate before use</td>
		</tr>
		<tr>
			<td>Ficoll-Paque PLUS</td>
			<td>GE Healthcare</td>
			<td>17-1440-02</td>
			<td></td>
		</tr>
		<tr>
			<td>gentamycin (10mg/ml)</td>
			<td>Gibco</td>
			<td>15710-064</td>
			<td></td>
		</tr>
		<tr>
			<td>Hema3 Staining Set</td>
			<td>Fisher</td>
			<td>122-911</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP310-500</td>
			<td></td>
		</tr>
		<tr>
			<td>hypoxanthine</td>
			<td>Sigma</td>
			<td>H9636</td>
			<td></td>
		</tr>
		<tr>
			<td>Ionomycin</td>
			<td>Sigma</td>
			<td>I3909</td>
			<td></td>
		</tr>
		<tr>
			<td>MEM non-essential amino acids (10mM)</td>
			<td>Gibco</td>
			<td>11140</td>
			<td></td>
		</tr>
		<tr>
			<td>PMA</td>
			<td>Sigma</td>
			<td>P8139</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 powder</td>
			<td>Life-Technologies</td>
			<td>31800-022</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI-1640 with L-glutamine and HEPES</td>
			<td>Thermo Scientific</td>
			<td>SH30255.01</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium bicarbonate (powder, cell culture)</td>
			<td>Sigma</td>
			<td>S5761</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Pyruvate (100mM)</td>
			<td>Gibco</td>
			<td>11360</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris (Trizma base)</td>
			<td>Sigma</td>
			<td>T6066</td>
			<td></td>
		</tr>
		<tr>
			<td>Trizol</td>
			<td>Ambion</td>
			<td>15596018</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan Blue (0.4%)</td>
			<td>Gibco</td>
			<td>15250-061</td>
			<td></td>
		</tr>
		<tr>
			<td>BD CompBead</td>
			<td>BD</td>
			<td>552843, 552845</td>
			<td>depends on antibodies used</td>
		</tr>
		<tr>
			<td>Parasite Gas Mixture</td>
			<td>By special order</td>
			<td></td>
			<td>3% CO2, 1% O2, balance N2</td>
		</tr>
	</tbody>
</table>

an in vitro model for measuring immune responses to malaria in the context of hiv co-infection

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.

The graphs depict the levels of IFN&#947; production from NKT cells (Figure 2), using CD56+CD3+&#947;&#948;- 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 &#947;&#948; 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 gra...

Watch this Scientific Journal Video about An In Vitro Model for Measuring Immune Responses to Malaria in the Context of HIV Co-infection at JoVE.com

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

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.

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

Malaria and HIV co-infection is a growing health priority. However, most research on malaria or HIV currently focuses on each infection individually. ...

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