Gammaherpesvirus Co-infection with Malaria Suppresses Anti-parasitic Humoral Immunity

<div><p>Immunity to non-cerebral severe malaria is estimated to occur within 1-2 infections in areas of endemic transmission for <i>Plasmodium falciparum</i>. Yet, nearly 20% of infected children die annually as a result of severe malaria. Multiple risk factors are postulated to exacerbate malarial disease, one being co-infections with other pathogens. Children living in Sub-Saharan Africa are seropositive for Epstein Barr Virus (EBV) by the age of 6 months. This timing overlaps with the waning of protective maternal antibodies and susceptibility to primary <i>Plasmodium</i> infection. However, the impact of acute EBV infection on the generation of anti-malarial immunity is unknown. Using well established mouse models of infection, we show here that acute, but not latent murine gammaherpesvirus 68 (MHV68) infection suppresses the anti-malarial humoral response to a secondary malaria infection. Importantly, this resulted in the transformation of a non-lethal <i>P</i>. <i>yoelii</i> XNL infection into a lethal one; an outcome that is correlated with a defect in the maintenance of germinal center B cells and T follicular helper (Tfh) cells in the spleen. Furthermore, we have identified the MHV68 M2 protein as an important virus encoded protein that can: (i) suppress anti-MHV68 humoral responses during acute MHV68 infection; and (ii) plays a critical role in the observed suppression of anti-malarial humoral responses in the setting of co-infection. Notably, co-infection with an M2-null mutant MHV68 eliminates lethality of <i>P</i>. <i>yoelii</i> XNL. Collectively, our data demonstrates that an acute gammaherpesvirus infection can negatively impact the development of an anti-malarial immune response. This suggests that acute infection with EBV should be investigated as a risk factor for non-cerebral severe malaria in young children living in areas endemic for <i>Plasmodium</i> transmission.</p></div

MHV68 and <i>Plasmodium</i> co-infection results in defective splenic T follicular helper (Tfh) response.

<p>The timeline and experimental set up was identical to that shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004858#ppat.1004858.g001" target="_blank">Fig 1A</a>. (A) Representative flow plots for gating strategies used to define the global Tfh population (CD4+ PD-1+ CXCR5+), germinal center Tfh (CD4+ GL7+ CXCR5+) and activated/antigen specific Tfh (CD4+ CD44+ PD-1+ CXCR5+). (B) Absolute values for all three Tfh subsets are plotted for the <i>P</i>. <i>yoelii</i> XNL (Day 23, all Tfh subsets, <i>P</i>. <i>yoelii</i> vs. co-infected, p<0.05 Mann Whitney U-test) or (C) <i>P</i>. <i>chabaudi</i> co-infection models at multiple time points (Day 23, all Tfh subsets, <i>P</i>. <i>chabaudi</i> vs. co-infected, p<0.05 Mann Whitney U-test).</p

Acute, but not latent, MHV68 infection results in suppressed humoral response.

<p>(A) Timeline of infection. C57BL/6 mice were infected with 1000 PFU of MHV68 IN at day -60, -30, -15 or -7 and challenged with 10⁵ pRBCs on day 0. Absolute number of (B) splenic GC B cell (B220+ GL7+ CD95+) and plasma cell (CD3- B220int CD138+) populations at day 16 post <i>P</i>. <i>yoelii</i> XNL infection (For GC and PC: Day -7 and Day -15 co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p<0.05/ Day -30 co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p>0.05). (C) MHV68 and <i>P</i>. <i>yoelii</i> XNL specific IgG responses at day 16 post <i>P</i>. <i>yoelii</i> XNL infection (Day -7 and Day -15 co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p<0.05/ Day -30 co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p>0.05). (D) Global Tfh population (CD4+ PD-1+ CXCR5+), germinal center Tfh (CD4+ GL7+ CXCR5+) and activated/antigen specific Tfh (CD4+ CD44+ PD-1+ CXCR5+) in the spleen at day 16 post <i>P</i>. <i>yoelii</i> XNL infection.</p

MHV68 co-infection with the non-lethal <i>P</i>. <i>yoelii</i> XNL in C57BL/6 results in lethal malarial disease and suppressed <i>Plasmodium</i> specific IgG response.

<p>(A) Timeline of infection. 6–8 week old C57BL/6 mice were infected with 1000 PFU of MHV68 on day -7 followed by infection with 10⁵pRBCs of non-lethal <i>P</i>. <i>yoelii</i> XNL or <i>P</i>. <i>chabaudi</i> AS. Infections consisted of 5 experimental groups: MHV68 + <i>Plasmodium</i>, <i>Plasmodium</i>, MHV68 or mock infected. Each experimental group consisted of n = 5 and was repeated twice. Animals were sacrificed at days 8, 12, 16 and 23 post <i>P</i>. <i>yoelii</i> XNL infection or day 7, 11, 15 and 23 post <i>P</i>. <i>chabaudi</i> AS infection for collection of spleen, lung and blood. (B) Survival analysis of animals co-infected with MHV68 and <i>P</i>. <i>yoelii</i> XNL or <i>P</i>. <i>chabaudi</i> AS. Total IgG and IgM levels in serum in (C) <i>P</i>. <i>yoelii</i> XNL (Day 23 IgG—<i>P</i>. <i>yoelii</i> vs co-infected: p<0.05 Mann Whitney U-test) or (D) <i>P</i>. <i>chabaudi</i> AS co-infection model (Day 11 IgG—<i>P</i>. <i>chabaudi</i> vs co-infected: p<0.05 Mann Whitney U-test). Parasite specific IgG levels in serum during (E) <i>P</i>. <i>yoelii</i> XNL (day 23 post infection, <i>P</i>. <i>yoelii</i> vs co-infected: p<0.05 Mann Whitney U-test) or (F) <i>P</i>. <i>chabaudi</i> AS (day 11 post infection, <i>P</i>. <i>chabaudi</i> vs co-infected: p<0.05 Mann Whitney U-test) co-infection.</p

The MHV68 M2 gene product is necessary for virus mediated humoral suppression and lethality during <i>Plasmodium</i> co-infection.

<p>(A) MHV68 specific IgG titers from serum of animals infected with the MR (M2.Marker Rescue) or M2.Stop (ST, M2-null) viruses. Serum was collected and analyzed on days 7, 14 and 21 post infection with either virus (n = 10/ virus) (Day 21, MR vs. M2.Stop, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p<0.05). (B) <i>P</i>. <i>yoelii</i> XNL specific IgG response during <i>P</i>. <i>yoelii</i> XNL co-infection with either the M2.MR or M2.Stop virus. Serum was collected at day 20 post infection with <i>P</i>. <i>yoelii</i> XNL (WT + <i>P</i>. <i>yoelii</i> co-infected vs. <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p<0.05/ WT + <i>P</i>. <i>yoelii</i> co-infected vs. M2.Stop + <i>P</i>. <i>yoelii</i>, Kruskal Wallis p<0.05; Dunn’s pairwise comparison test p>0.05). (C) Survival curve during <i>P</i>. <i>yoelii</i> XNL co-infection with either the M2.MR or M2.Stop virus. <u>Note</u>: data representing <i>P</i>. <i>yoelii</i> XNL + MHV68 co-infection is the identical data set to that in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004858#ppat.1004858.g001" target="_blank">Fig 1B</a>. It was added in panel C for comparative purposes. (D) % parasitemia in the periphery during <i>P</i>. <i>yoelii</i> XNL, <i>P</i>. <i>yoelii</i> XNL +MR and <i>P</i>. <i>yoelii</i> XNL + M2.Stop infection.</p

MHV68 suppresses splenic B cell responses during co-infection with <i>Plasmodium</i>.

<p>The timeline and experimental set up was identical to that shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1004858#ppat.1004858.g001" target="_blank">Fig 1A</a>. (A) Absolute numbers of splenic GC B cell populations (B220+ GL7+ CD95+) during <i>P</i>. <i>yoelii</i> XNL and <i>P</i>. <i>chabaudi</i> AS co-infection models with representative gating strategy (Day 12 post <i>P</i>. <i>yoelii</i> or Day 15 post <i>P</i>. <i>chabaudi</i>; <i>Plasmodium</i> vs. co-infected, p<0.05, Mann Whitney U-test). (B) Absolute numbers of splenic plasma cell populations (CD3- B220int CD138+) during <i>P</i>. <i>yoelii</i> XNL AND <i>P</i>. <i>chabaudi</i> AS co-infection models with representative gating strategy (Day 12 post <i>P</i>. <i>yoelii</i> or Day 11 post <i>P</i>. <i>chabaudi</i>; <i>Plasmodium</i> vs. co-infected, p<0.05, Mann Whitney U-test). (C) Spleen section for mock infected, MHV68 infected, <i>P</i>. <i>yoelii</i> XNL infected and MHV68 and <i>P</i>. <i>yoelii</i> XNL co-infected animals at day 8 post infection with <i>P</i>. <i>yoelii</i> XNL (or day 15 post-infection with MHV68). Green: B220-FITC (B cells), Blue: GL7-AF660 (Germinal center B cells) and Red: CD3-AF568 (T cells).</p