Longevity and Composition of Cellular Immune Responses Following Experimental Plasmodium falciparum Malaria Infection in Humans

Cellular responses to Plasmodium falciparum parasites, in particular interferon-gamma (IFNγ) production, play an important role in anti-malarial immunity. However, clinical immunity to malaria develops slowly amongst naturally exposed populations, the dynamics of cellular responses in relation to exposure are difficult to study and data about the persistence of such responses are controversial. Here we assess the longevity and composition of cellular immune responses following experimental malaria infection in human volunteers. We conducted a longitudinal study of cellular immunological responses to sporozoites (PfSpz) and asexual blood-stage (PfRBC) malaria parasites in naïve human volunteers undergoing single (n = 5) or multiple (n = 10) experimental P. falciparum infections under highly controlled conditions. IFNγ and interleukin-2 (IL-2) responses following in vitro re-stimulation were measured by flow-cytometry prior to, during and more than one year post infection. We show that cellular responses to both PfSpz and PfRBC are induced and remain almost undiminished up to 14 months after even a single malaria episode. Remarkably, not only ‘adaptive’ but also ‘innate’ lymphocyte subsets contribute to the increased IFNγ response, including αβT cells, γδT cells and NK cells. Furthermore, results from depletion and autologous recombination experiments of lymphocyte subsets suggest that immunological memory for PfRBC is carried within both the αβT cells and γδT compartments. Indeed, the majority of cytokine producing T lymphocytes express an CD45RO+ CD62L- effector memory (EM) phenotype both early and late post infection. Finally, we demonstrate that malaria infection induces and maintains polyfunctional (IFNγ+IL-2+) EM responses against both PfRBC and PfSpz, previously found to be associated with protection. These data demonstrate that cellular responses can be readily induced and are long-lived following infection with P. falciparum, with a persisting contribution by not only adaptive but also (semi-)innate lymphocyte subsets. The implications hereof are positive for malaria vaccine development, but focus attention on those factors potentially inhibiting such responses in the field

Detection of anti-N antibodies by ELISA.

<p>Sera were obtained 7, 14 and 21 days post vaccination (DPV) and at 0, 7, 14 and 21 days post challenge (DPC). Titers are expressed as percentage competition ratio of the optical densities (OD) of the sample and the OD of the negative control (%S/N). All values lower than 40% are considered positive, between 40–50% are considered doubtful and above 50% are considered negative. The 40% and 50% cut-offs are represented by solid and interrupted lines, respectively. Results obtained from analysis of each individual animal from the mock-group (C1–C8), low-dose group (L1–L8), medium-dose group (M1–M8) or high-dose group (H1–H7) are depicted.</p

Detection of viral RNA in plasma by qRT-PCR.

<p>Plasma samples were collected daily at the first 7 days post challenge (DPC) and subsequently on DPC 9, 11, 14 and 21. Viral RNA copy numbers detected in individual animals of the mock-vaccinated group (C1–C8), low-dose group (L1–L8), medium-dose group (M1–M8) and high-dose group (H1–H7) are depicted.</p

Virus neutralization test (VNT).

<p>Sera were obtained from lambs of the mock group, low-dose group, medium-dose group and high-dose group. The white bars represent VNT titers determined 21 days post vaccination (DPV) and the black bars represent the VNT titers determined 21 days post challenge (DPC). Results obtained from analysis of each individual animal from the mock-group (C1–C8), low-dose group (L1–L8), medium-dose group (M1–M8) and high-dose group (H1–H7) are depicted. The detection limit of the assay is represented by an interrupted line. Lamb C3 died 7 days after challenge, therefore no serum sample was collected at 21 DPC.</p

Construction of the S-Gn segment and expression of Gn.

<p>(A) Schematic representation of the S segment of RVFV (upper panel) and the S segment in which the NSs gene is replaced for the codon-optimized Gn gene (lower panel). Distribution of Gn in Rep-Gn cells (B and E) and in BHK cells infected with NSR-Gn at an MOI of 0.5 (C and F). Panels D and G represent BHK control cells. Upper panels represent permeabilized cells and lower panels represent nonpermeabilized cells. The cells were stained with an anti-Gn monoclonal antibody and a Texas Red-labeled secondary antibody. Nuclei were visualized by DAPI staining.</p

Rectal temperatures of vaccinated and mock-vaccinated lambs before and after challenge with RVFV.

<p>Fever was defined as a rectal body temperature above 40.5°C (interrupted line). Body temperatures of mock-vaccinated lambs (C1–C8) and lambs vaccinated with a low dose (L1–L8), medium dose (M1–M8) or high dose (H1–H7) of NSR-Gn are depicted individually.</p

Humoral and cellular immune responses elicited by vaccination of mice with NSR or NSR-Gn.

<p>(A) VNT titers in sera collected from mice vaccinated with NSR or NSR-Gn before vaccination (DPV -1) and 13, 22 and 29 DPV. Bars represent average titers (n = 6) of each group with standard deviation. The detection limit of the assay is depicted by the interrupted line. (B) Detection of IFN-γ-producing splenocytes isolated from mice vaccinated with NSR or NSR-Gn. Splenocytes were isolated and seeded at a density of 5×10⁵ cells/well in triplicate and stimulated for 12 hours with the indicated peptides or the ectodomain of Gn. Bars represent an average number of IFN-γ producing cells (n = 4) per group with standard deviation. The non-parametric Mann-Whitney test was used for statistical analysis and statistical significance between the groups is depicted by asterisks (*p<0.05; **p<0.01).</p

Surface expression of CD40, CD80, CD83, CD86, MHC-I and MHC-II on DCs at 24 h after NSR infection as measured by flow cytometry.

<p>Immature DCs were infected with NSR, mock-infected with NSRmock, or stimulated with LPS (left and middle panels). Alternatively, cells were infected with NSR or mock-infected with NSRmock in the presence of LPS (right panels). The left panel shows representative histograms of surface marker measurements on cells stimulated with LPS, mock infected cells (NSRmock), cells infected with NSR (GFP+) and uninfected bystander DCs (GFP-). Expression of markers in untreated cells and an irrelevant isotype control are depicted. The middle and right panels represent average data from 4 independent experiments performed with cells from 3 donors. The box plots depict MFI of the different markers relative to untreated cells. A black asterisk indicates upregulation compared to the control and a red asterisk indicates downregulation.</p