Chronic schistosomiasis suppresses HIV-specific responses to DNA-MVA and MVA-gp140 Env vaccine regimens despite antihelminthic treatment and increases helminth-associated pathology in a mouse model

<div><p>Future HIV vaccines are expected to induce effective Th1 cell-mediated and Env-specific antibody responses that are necessary to offer protective immunity to HIV infection. However, HIV infections are highly prevalent in helminth endemic areas. Helminth infections induce polarised Th2 responses that may impair HIV vaccine-generated Th1 responses. In this study, we tested if <i>Schistosoma mansoni</i> (Sm) infection altered immune responses to SAAVI candidate HIV vaccines (DNA and MVA) and an HIV-1 gp140 Env protein vaccine (gp140) and whether parasite elimination by chemotherapy or the presence of Sm eggs (SmE) in the absence of active infection influenced the immunogenicity of these vaccines. In addition, we evaluated helminth-associated pathology in DNA and MVA vaccination groups. Mice were chronically infected with Sm and vaccinated with DNA+MVA in a prime+boost combination or MVA+gp140 in concurrent combination regimens. Some Sm-infected mice were treated with praziquantel (PZQ) prior to vaccinations. Other mice were inoculated with SmE before receiving vaccinations. Unvaccinated mice without Sm infection or SmE inoculation served as controls. HIV responses were evaluated in the blood and spleen while Sm-associated pathology was evaluated in the livers. Sm-infected mice had significantly lower magnitudes of HIV-specific cellular responses after vaccination with DNA+MVA or MVA+gp140 compared to uninfected control mice. Similarly, gp140 Env-specific antibody responses were significantly lower in vaccinated Sm-infected mice compared to controls. Treatment with PZQ partially restored cellular but not humoral immune responses in vaccinated Sm-infected mice. Gp140 Env-specific antibody responses were attenuated in mice that were inoculated with SmE compared to controls. Lastly, Sm-infected mice that were vaccinated with DNA+MVA displayed exacerbated liver pathology as indicated by larger granulomas and increased hepatosplenomegaly when compared with unvaccinated Sm-infected mice. This study shows that chronic schistosomiasis attenuates both HIV-specific T-cell and antibody responses and parasite elimination by chemotherapy may partially restore cellular but not antibody immunity, with additional data suggesting that the presence of SmE retained in the tissues after antihelminthic therapy contributes to lack of full immune restoration. Our data further suggest that helminthiasis may compromise HIV vaccine safety. Overall, these findings suggested a potential negative impact on future HIV vaccinations by helminthiasis in endemic areas.</p></div

<i>N. brasiliensis</i> induced smooth muscle cell hypertrophy/hyperplasia is unaffected in iLck^creIL-4Rα^−/lox mice.

<p>Haematoxylin and eosin stained sections were used to determine the smooth muscle cell layer thickness from Day 3, 7 and 10 <i>N. brasiliensis-</i>infected iLck^creIL-4Rα^−/lox and control mice. Representative photomicrographs are shown from control mice at days 3, 7 and 10 at 40× magnification. Also shown is a photomicrograph at 200× showing the longitudinal and circular smooth muscle layers included in the measurement (A). Measurements are shown in a bar graph (B) with mean values+SEM and represent 2 independent experiments with n = 4 or 5 mice per group. Ns = not significant. One-Way-ANOVA, ***<i>P<.001</i>.</p

IL-4 responsive T cells are not needed for expulsion of <i>N. brasiliensis.</i>

<p>iLck^creIL-4Rα^−/lox and control mice were infected with 750 <i>N. brasiliensis</i> L3 larvae. Faeces were collected from day 6 to 14 post infection (PI) and egg production was calculated using the modified McMaster technique (A). At days 7 and 10 PI the worm burden in the small intestine was assessed (pooled from 3 experiments) (B). Intestinal goblet cell hyperplasia was assessed by determining the total number of PAS-positive goblet cells per 5 villi in histological sections of the small intestine at day 7 and 10 PI (C). Mucus and PAS staining at days 7 and 10 PI. Representative photomicrographs are shown from individual mice and <i>N. brasiliensis</i> is indicated with a black arrow (D). Total IgE production in the serum was measured by ELISA at day 7 and 10 PI (E). The graphs show mean values ± SEM and represent the results of three independent experiments, except B and E where 2–3 independent experiments were combined with n = 4 or 5 mice per group. ND, not detected. One-Way-ANOVA, *<i>P<.05</i>, **<i>P<.01</i>, ***<i>P<.001</i> for all experiments.</p

Reduced IL-4 response in <i>N. brasiliensis-</i>infected iLck^creIL-4Rα^−/lox and IL-4Rα^−/− mice.

<p>Mice were infected with 750 <i>N. brasiliensis</i> L3 larvae and at days 7 and 10 PI CD4⁺ cells from pooled mesenteric lymph nodes were isolated by negative selection (purity>90%) then restimulated with anti-CD3 for 48 hours and IL-4, IL-13, INF-γ, IL-17 cytokine concentration of the supernatant determined by ELISA (A). Further, IL-4 and IL-13 concentrations were determined in homogenates of the jejunum (B). The graphs show mean values+SEM and are representative of the results of three independent experiments with IL-17 only determined in one experiment for CD4⁺ T cells and IL-13 in two independent experiments for homogenates, with n = 4 or 5 per group. One-Way-ANOVA, *<i>P<.05</i>, **<i>P<.01</i>, ***<i>P<.001</i>.</p

Cellular and antibody responses to HIV vaccines in SmE inoculated mice.

<p>Spleens and blood were collected 12 days after the last vaccination. Splenocytes were stimulated the induced cytokines were analysed in an IFN-γ (A) and IL-2 (B) ELISpot, cytokine bead array (CBA) (C, D, and E) assays. The individual bars represent the magnitudes of the cumulative cytokine levels. HIV-1 Env gp140-specific IgG1, IgG2a and IgG2b antibodies were analysed in the sera by an ELISA (F). Values are expressed as antibody units and the mean (AUs) for the 8–12 animals in each group shown as a horizontal bar. An IFN-γ:IL-4 ratio (G) was calculated from the CBA data. Results represent 3 independent experiments. Statistical analysis was performed using unpaired, two-tailed t-test analysis. (*: p<0.05; **: p<0.01).</p

Th1/Th2 profile: <i>S</i>. <i>mansoni</i> infection induces a strong Th2 biased cellular and antibody responses.

<p>Spleens and blood were obtained from vaccinated Sm-free (blue) and Sm-infected (red) mice 16 weeks post infection. Splenocytes were prepared and stimulated with 1 μg/ml Con A for 48 (A) and 23 (B) hours and cytokines were measured in cytometric bead array and ELISpot assays respectively. Other splenocytes were stimulated with PMA/Ionomycin for 6hrs (C) and the induced intracellular cytokines were measured by flow cytometry. Total Th1 and Th2 antibodies were also measured in sera using an antibody ELISA (D). Results represent 3 independent experiments and plotted as the mean + SEM. Statistical analysis was performed using unpaired, two-tailed t-test analysis followed by FDR for multiple comparisons. (*: p<0.05; **: p<0.01; ***: p<0.001).</p

Immunasation schedule for live <i>S</i>. <i>mansoni</i> cercaria infection groups.

<p>Immunasation schedule for live <i>S</i>. <i>mansoni</i> cercaria infection groups.</p

Immunisations schedule for <i>S</i>. <i>mansoni</i> egg challenge groups.

<p>Immunisations schedule for <i>S</i>. <i>mansoni</i> egg challenge groups.</p

Analysis of livers and spleens of mice after vaccination with a DNA+MVA vaccine regimen.

<p>Groups of mice were infected with 30–35 Sm cercariae. Some groups were treated twice with PZQ after 8.5 weeks post infection. Vaccination with a DNA+MVA vaccine regimen was performed as shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1007182#ppat.1007182.t001" target="_blank">Table 1</a>. Spleens and livers were harvested 12 days after last vaccination, weighed and prepared for various analyses. (A): Representative histological micrographs showing liver granulomas after staining with either H&E or CAB; (B): Granuloma sizes; (C) Liver weights; (D): Spleen weights; (E): hydroxyproline content and (F): Number of eggs per gram of liver. For Figs B-D, the results represent 3 independent experiments. For Figs E and F, the data is representative of 2 independent experiments. Means are shown as horizontal bars. Statistical analysis was performed using unpaired, two-tailed t-test analysis followed by FDR for multiple comparisons. (*: p>0.05; **: p<0.01; ***: p<0.001).</p

Sm infection alters vaccine-specific cellular responses and treatment with PZQ only partially restores these responses close to normal.

<p>Mice were chronically infected with Sm before vaccination with a DNA-vectored and MVA-vectored HIV-1 (DNA+MVA) vaccine or an MVA-vectored HIV-1 and a HIV-1 gp140 Env protein (MVA+gp140) vaccine regimen with or without prior anti-helminthic treatment with PZQ. Spleens were collected 12 days after the last vaccination. The splenocytes were stimulated and the induced cytokines measured in an IFN-γ (A) and IL-2 (B) ELISpot, cytometric bead array (C, D, and E), and intracellular cytokine staining (F, G, and H) assays. Results represent 3 independent experiments and plotted as the mean + SEM. Statistical analysis was performed using unpaired, two-tailed t-test analysis followed by FDR for multiple comparisons. (*: p<0.05; **: p<0.01; ***: p<0.001).</p