Disruption of antigenic variation proves crucial for effective parasite vaccine. Supplementary Results

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1 Disruption of antigenic variation proves crucial for effective parasite vaccine Fernando D. Rivero, Alicia Saura, Cesar G. Prucca, Pedro G. Carranza, Alessandro Torri, and Hugo D. Lujan Supplementary Results Table S1 Expression of different VSPs on the surface of transgenic trophozoites. Reactivity of Giardia trophozoites expressing a particular VSP to different VSP mabs, as determined by indirect immunofluorescence assays using specific monoclonal antibodies. Anti-VSP antibodies used were: VSP9B10, mab 9B10; VSP1267, mab 5C1; VSPA6, mab 6A7; VSPS1, mab 1B2; VSPS2, mab7b8; VSPS7, mab6f8; VSPH7, mab G10/4; VSPS12, mab 7G8; VSPS6, mab 1B4. Anti-BiP (mab 9C9) served as a negative control. Samples included mock-transfected WB9B10 trophozoites (WB9B10) or trophozoites transfected with the antisense construct of genes encoding RdRP (RAS) and Dicer (DAS) after 12 h in culture. Goat anti-mouse immunoglobulins were used as control (Control). Figures represent the mean value of three independent experiments performed in duplicate expressed as percentage of reactivity. Results indicated that many different VSPs are expressed in single trophozoites, as judged by addition of each percentage. mab anti-bip produced no reaction in non-permeabilised trophozoites. VSPH7 from the GS-M isolate is not expressed in the WB isolate. mab WB9B10 DAS RAS DAS + RAS Control B C E G10/ C B B F G B

2 Tables S2-S4. Challenge infection using trophozoites or cysts of gerbils previously infected with different trophozoite populations. Gerbils were infected by orogastric inoculation of PBS (None) or trophozoites expressing VSP9B10 (WB9B10), VSP1267 (WB1267), VSPA6 (WBA6), or transgenic Giardia in which Dicer (DAS) or RdRP (RAS) have been silenced, as well as a 1:1 mixture of both (DAS + RAS). Two months post-inoculation, half of the animal groups were treated with Metronidazole (MNZ). Two, four, and 12 months post-inoculation gerbils were challenged with a clonal population of trophozoites expressing VSP9B10 (Table S2), VSP1267 (Table S3), or cysts obtained from stool samples of infected animals (Table S4). Infection was verified by the presence of cysts in stool samples and, in few cases, by killing selected gerbils (k) to determine the presence or not of trophozoites in the small intestine. Infected animals are indicated by a number followed by a (+) symbol, while no infection is indicated with a (-) symbol. Percentage of infected animals is shown between parentheses. If an animal died during the experiment a number followed by a (d) is indicated. No differences were noticed between treated or non-treated animals. Primary Infection with Table S2: Challenge infection with trophozoites of clone WB9B10 Number of gerbils MNZ month 2 month 4 month 12 None 30 Yes 30+ (100%) N/A N/A None 30 No 30+ (100%) N/A N/A WB9B10 30 Yes 30- (0%) N/A N/A WB9B10 30 No (3.3%) N/A N/A WB Yes 30+ (100%) N/A N/A WB No 29+ 1k (100%) N/A N/A WBA6 30 Yes 30+ (100%) N/A N/A WBA6 30 No 30+ (100%) N/A N/A Dicer-AS 90 Yes (10%) 30- (0%) 30- (0%) Dicer-AS 90 No 29-1k (0%) 28-2k (0%) 27-3k (0%) 90 Yes (3.3%) 29-1k (0%) 29-1k (0%) 90 No (6.6%) 28-2k (0%) 29-1k (0%) Dicer-AS & 90 Yes k (3.3%) 30- (0%) (3.3%) Dicer-AS & 90 No 29-1k (0%) (3.3%) 30- (0%) 2

3 Primary Infection with Table S3: Challenge infection with trophozoites of clone WB1267 Numbe r of gerbils MNZ month 2 month 4 month 12 None 30 Yes 30+ (100%) N/A N/A None 30 No 30+ (100%) N/A N/A WB9B10 30 Yes 30+ (100%) N/A N/A WB9B10 30 No 30+ (100%) N/A N/A WB Yes 30- (0%) N/A N/A WB No (3.3%) N/A N/A WBA6 30 Yes 30+ (100%) N/A N/A WBA6 30 No 30+ (100%) N/A N/A Dicer-AS 90 Yes (6.6%) k (3.3%) 30- (0%) Dicer-AS 90 No (3.3%) 29-1d (0%) 29-1k (0%) 90 Yes (3.3%) (3.3%) (3.3%) 90 No k (3.3%) 28-2k (0%) 30- (0%) Dicer-AS & 90 Yes (6.6%) (3.3%) 29-1k (0%) Dicer-AS & 90 No 30- (0%) 30- (0%) 30- (0%) Primary Infection with Table S4: Protection against challenge infection with cysts Numbe r of gerbils MNZ month 2 month 4 month 12 None 30 Yes 30+ (100%) N/A N/A None 30 No 30+ (100%) N/A N/A WB9B10 30 Yes 30+ (100%) N/A N/A WB9B10 30 No 29+ 1k (100%) N/A N/A WB Yes 30+ (100%) N/A N/A WB No 29+ 1k (96.6%) N/A N/A WBA6 30 Yes 30+ (100%) N/A N/A WBA6 30 No 30+ (100%) N/A N/A Dicer-AS 90 Yes 30- (0%) 30- (0%) 30- (0%) Dicer-AS 90 No 29-1k (0%) 30- (0%) 29-1k (0%) 90 Yes (3.3%) 29-1k (0%) 29-1k (0%) 90 No (3.3%) k (3.3%) 29-1k (0%) Dicer-AS & 90 Yes (3.3%) 30- (0%) 30- (0%) Dicer-AS & 90 No (3.3%) (3.3%) 29-1k (0%) 3

4 Supplementary Fig. S1. Challenge inoculation of gerbils with trophozoites expressing a particular VSP following primary infection with Giardia expressing the entire set of VSPs. Gerbils previously infected with clones WB9B10, WB1267, DAS, or RAS (a-d) were challenged with clonal population of clones WB9B10 (e-h), WB1267 (i-l), or purified cysts (m-p). Figure shows representative images of immunofluorescence assays on gerbil stool samples using the FITC-conjugated mab anti-cwp2 (green). Animals previously infected with clone WB9B10 were refractory to subsequent infection with the same clone (no cysts are observed in the faeces) but readily infected by trophozoites expressing VSP1267 (high number of cysts are easily detected). Gerbils previously infected with DAS or RAS were highly protected to subsequent infections with clones WB9B10 or WB1267 (no cysts could be found in these stool samples). 4

5 Supplementary Fig. S2 VSPs are resistant to proteolytic digestion. Monoclonal antibodies against two particular VSPs (mab G10/4, which recognize a conformational epitope in VSPH7 of the GS/M isolate, and mab 9B10, which detect a non-conformational epitope in VSP9B10 of the WB isolate) were used to test their reactivity to VSPs on clonal trophozoite populations by indirect immunofluorescence assays. Parasites resuspended in PBS at ph 7.4 were treated for 90 min with variable concentrations of trypsin similar of those found in the upper small intestine, washed twice with PBS, and incubated for 60 min at 4 with the corresponding mab. In all cases the epitope remains intact as determined by immunofluorescence microscopy. The antibodies agglutinate the trophozoites and label the surface of the parasites. PC: Phase Contrast. IFA: Immunofluorescence Assay. Trypsin concentration WB-9B10 GS/M-H7 PC IFA PC IFA 100 µg/ml 150 µg/ml 200 µg/ml 5

6 Supplementary Fig. S3. VSPs are resistant to variable phs. Monoclonal antibodies against two particular VSPs (mab G10/4, which recognize a conformational epitope in VSPH7 of the GS/M isolate, and mab 9B10, which detect a non-conformational epitope in VSP9B10 of the WB isolate) were used to test their reactivity to VSPs on clonal trophozoite populations by indirect immunofluorescence assays. Parasites were resuspended in RPMI at variable phs for 90 min, washed twice with PBS, and incubated for 60 min at 4 with the corresponding mab. In all cases the epitope remains intact as determined by immunofluorescence microscopy. The antibodies agglutinate the trophozoites and label the surface of the parasites. PC: Phase Contrast. IFA: Immunofluorescence Assay. ph WB-9B10 GS/M-H7 PC IFA PC IFA

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8 Supplementary Fig. S4. Preparation of antigens for immunization. A monoclonal antibody against the conserved cytoplasmic tail of all VSPs was utilized to immunopurify VSPs from trophozoites in which Giardia Dicer has been knocked-down (left), the mab 9B10 was utilized to immunopurify the VSP9B10 from wild type WB9B10 trophozoites (centre), and a monoclonal antibody anti-ha was used to immunopurify the molecular chaperone GPR78/BiP with an HA-tag from trophozoites of clone WB9B10 over-expressing this molecule (right). The protein mixtures were then analyzed by electrophoresis and subsequent labelling with Silver Stain (SS) and detected by western blotting (WB). Molecular weight markers are at the left. 8

9 Tables S5-S7. Challenge infection using trophozoites or cysts of gerbils previously immunized with different VSPs or VSP mixtures. Gerbils were infected by orogastric inoculation of PBS (None) or purified VSP9B10, VSP1267, VSPA6, or VSPs purified from transgenic Giardia in which Dicer (DAS) or RdRP (RAS) have been knocked-down, as well as a 1:1 mixture of both (DAS + RAS). Two months post-inoculation, half of the animal groups were treated with Metronidazole (MNZ) to avoid any possible contamination of the animals. Two, four, and 12 months post-immunization, gerbils were challenged with a clonal population of trophozoites expressing VSP9B10 (Table S5), VSP1267 (Table S6), or cysts obtained from stool samples of infected animals (Table S7). Infection was verified for the presence of cysts in stool samples and, in few cases, by killing selected gerbils (k) to determine the presence or not of trophozoites in the small intestine. Infected animals are indicated by a number followed by a (+) symbol, while no infection is indicated with a (-) symbol. Percentage of infected animals is shown between parentheses. If an animal died during the experiment a number is followed by a (d) is indicated. N/A means not applicable. No differences were noticed between treated or non-treated animals. Immunization with VSPs or BiP Table S5: Challenge infection with trophozoites of clone WB9B10 Number of gerbils MNZ month 2 month 4 month 12 None 30 Yes 30+ (100%) N/A N/A None 30 No 30+ (100%) N/A N/A WB9B10 30 Yes 30- (0%) N/A N/A WB9B10 30 No (6.6%) N/A N/A WB Yes 30+ (100%) N/A N/A WB No 30+ (100%) N/A N/A WBA6 30 Yes 30+ (100%) N/A N/A WBA6 30 No 30+ (100%) N/A N/A Dicer-AS 90 Yes (16.6%) (10%) k (3.3%) Dicer-AS 90 No k (10%) k (3.3%) (10%) 90 Yes (6.6%) d (3.4%) k (3.3%) 90 No k (6.6%) k (6.6%) 30- (0%) Dicer-AS & 90 Yes (3.3%) (3.3%) 30- (0%) Dicer-AS & 90 No 29-1k (0%) (3.3%) 29-1k (0%) BiP 30 Yes 30+ (100%) N/A N/A BiP 30 No 30+ (100%) N/A N/A 9

10 Immunization with VSPs or BiP Table S6: Challenge infection with trophozoites of clone WB1267 Number of gerbils MNZ month 2 month 4 month 12 None 30 Yes 30+ (100%) N/A N/A None 30 No 30+ (100%) N/A N/A WB9B10 30 Yes 30+ (100%) N/A N/A WB9B10 30 No 30+ (100%) N/A N/A WB Yes 30- (0%) N/A N/A WB No (3.3%) N/A N/A WBA6 30 Yes 30+ (100%) N/A N/A WBA6 30 No 29+ 1k (100%) N/A N/A Dicer-AS 90 Yes (3.3%) k (6.6%) (3.3%) Dicer-AS 90 No (10%) k (10%) (3.3%) 90 Yes (6.6%) (3.3%) 28-2k (0%) 90 No k (10%) (3.3%) 30- (0%) Dicer-AS & 90 Yes (3.3%) (3.3%) 29-1k (3.3%) Dicer-AS & 90 No 29-1k (0%) (3.3%) (3.3%) BiP 30 Yes 30+ (100%) N/A N/A BiP 30 No 30+ (100%) N/A N/A Immunization with VSPs or BiP Table S7: Protection against challenge infection with cysts Number of gerbils MNZ month 2 month 4 month 12 None 30 Yes 30+ (100%) N/A N/A None 30 No 30+ (100%) N/A N/A WB9B10 30 Yes 30+ (100%) N/A N/A WB9B10 30 No 29+ 1k (100%) N/A N/A WB Yes 30+ (100%) N/A N/A WB No 30+ (100%) N/A N/A WBA6 30 Yes (96.6%) N/A N/A WBA6 30 No 30+ (100%) N/A N/A Dicer-AS 90 Yes (10%) (10%) (6.6%) Dicer-AS 90 No k (10%) k (10%) 30- (0%) 90 Yes 30- (0%) k (3.3%) (3.3%) 90 No (6.6%) k (6.6%) (3.3%) Dicer-AS & 90 Yes (3.3%) (3.3%) 29-1k (0%) Dicer-AS & 90 No (3.3%) 29-1k (0%) 29-1k (0%) BiP 30 No 30+ (100%) N/A N/A BiP 30 No 30+ (100%) N/A N/A 10

11 Supplementary Fig. S5. Sera of infected human patients agglutinate the cells and recognize the surface of the trophozoites. Sera from 5 infected human patients and a newborn Giardia naïve individual, used as control, were directly-labelled with FITC and used to test their reactivity to VSPs on transgenic and wild type trophozoite populations by indirect immunofluorescence assays. Parasites were resuspended in culture medium and incubated for 60 min at 4 with the corresponding serum. WB-DAS (A), WB-9B10 (B), and GS/M-H7 (C) trophozoite populations were used. The sera from infected individuals agglutinate the trophozoites and label the surface of the parasites in contrast to a noninfected newborn used as control. PC: Phase Contrast. IFA: Immunofluorescence Assay. A WB-DAS Serum Agglutination PC IFA 1 98% 2 97% 3 98% 4 94% 5 90% Control Newborn serum 1% PBS 1% 11

12 B WB-9B10 Serum Agglutination PC IFA 1 87% 2 80% 3 75% 4 68% 5 91% Control Newborn Serum 1% PBS 1% 12

13 C GS/M-H7 Serum Agglutination PC IFA 1 67% 2 73% 3 88% 4 59% 5 83% Control Newborn Serum 1% PBS 1% 13