Nucleic acid receptor ligands improve vaccination efficacy against the filarial nematode Litomosoides

32 Infections with helminths affect more than one billion people worldwide. Despite an urgent 33 need there is no vaccine available that would confer long lasting protection against helminth 34 infections. Previous studies indicated that a vaccination with irradiated infective L3 reduces 35 the worm load. This present study investigated whether the additional activation of cytosolic 36 nucleic acid receptors as adjuvant improves the efficacy of a vaccination with irradiated L3 37 larvae of the rodent filaria Litomosoides sigmodontis . Subcutaneous injection of irradiated L3 38 larvae in combination with poly(I:C) or 3pRNA resulted in increased neutrophil recruitment to 39 the skin, accompanied by higher IP-10/CXCL10 and IFN-β RNA levels at the site of injection. 40 To investigate Serum analysis infection confirmed induction L. sigmodontis antibodies to the immunization serum larval motility vitro naïve cells. days after the challenge irradiated poly(I:C) cytosolic RNA receptor agonists.

(which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.11.499516 doi: bioRxiv preprint changes in the immune cell composition within the pleural cavity.

198
Immunization with adjuvants enhances functional parasite-specific antibody responses 199 Since the treatment with poly(I:C) or 3pRNA enhanced filarial clearance, the agonists were 200 included to the L. sigmodontis immunization strategy. To that end mice were subcutaneously 201 immunized with att. L3 larvae in combination with poly(I:C) or 3pRNA every two weeks for a 202 total of three times ( Figure 4A). This was followed by a natural challenge infection two weeks 203 after the last immunization. The serum obtained two weeks after full immunization, but prior to 204 challenge infection, was analyzed for parasite-specific antibodies. Immunization with att. L3 205 larvae alone or along with poly(I:C) or 3pRNA induced the production of L. sigmodontis-specific 206 IgE compared to non-immunized mice ( Figure 4B). All immunization regimes resulted in 207 significant production of L. sigmodontis-specific IgG1 ( Figure 4C). The immunization with att.

209
However, there was a significant IgG2a/b response in animals immunized with a combination 210 of att. L3 with non-formulated poly(I:C), poly(I:C) or 3pRNA. The use of non-formulated 211 poly(I:C) or poly(I:C) as adjuvant also significantly increased the IgG2a/b production compared 212 to the immunization with att. L3 larvae alone.

213
In order to assess functionality, an antibody-dependent cellular cytotoxicity assay was 214 performed. Since the immune cell counts of the naïve pleural cavity is too low to perform ex 215 vivo assays, naïve peritoneal cells, mainly consisting of myeloid and B cells (S2 Fig), were co-216 cultured with L. sigmodontis L3s, the medium was supplemented with the serum of immunized 217 mice and the motility of L3s was scored over time ( Figure 4E). The supplementation with serum 218 of unimmunized mice led to a continuous slow motility of L3s (score 3) after one day, and 219 discontinuous movements in part restricted to the ends of the L3s at two and three days of 220 culture (score 1.3, Figure 4F). The supplementation with serum from mice immunized with att.

221
L3 larvae alone resulted in significantly reduced larvae motilities on day one (score 2.4) and 222 three (score 0.8) of culture. Compared to this, the serum of animals immunized with a . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.11.499516 doi: bioRxiv preprint 9 223 combination of att. L3 larvae and non-formulated poly(I:C), poly(I:C) or 3pRNA resulted in 224 significantly reduced larval motility on day one (score 0.9 for non-formulated poly(I:C), score 2 225 for poly(I:C), score 1.8 for 3pRNA). On day three only the group with att. L3 and poly (I:C) 226 immunized serum had a motility score that was significantly lower (score 0.4) compared to the 227 effect of serum from mice immunized with att. L3 alone. In co-cultures with serum from animals 228 immunized with att. L3 larvae and non-formulated poly(I:C) the larval motility recovered to a 229 score of 2.1. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 12, 2022.

269
. CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.11.499516 doi: bioRxiv preprint 11 270 Despite the reduction in worm burden there were no notable changes in the pleural cell count 271 in any immunized group compared to the control animals ( Figure 5B)

295
In this study we demonstrate that type I interferon is increasingly produced during infection 296 with L. sigmodontis at the site of the parasite. The subcutaneous injection of att. L3 larvae 297 along with nucleic acid receptor agonists poly(I:C) and 3pRNA enhanced local immune . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 12, 2022. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 12, 2022. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.11.499516 doi: bioRxiv preprint reporter mice were provided by Prof. Dr. Eicke Latz (Institute of Innate Immunity, University   382 Hospital Bonn) and TLR7 -/-, TLR9 -/-, MDA5 -/as well as STING -/mice were provided by Prof. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.11.499516 doi: bioRxiv preprint Aidenbach, Germany), 2 mM EDTA (Carl Roth, Karlsruhe, Germany)) and 1 x 10 6  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.11.499516 doi: bioRxiv preprint to the manufacturer's protocol at an N/P ratio of 8.

465
Attenuation of third stage larvae (L3s) by irradiation was performed at the department of (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 12, 2022. (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.11.499516 doi: bioRxiv preprint filariaLitomosoides sigmodontisin BALB/c mice; comparative susceptibility of nine other inbred . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 12, 2022. . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.11.499516 doi: bioRxiv preprint 48. Volkmann L, Bain O, Saeftel M, Specht S, Fischer K, Brombacher F, Matthaei KI, Hoerauf

750
A. Murine filariasis: interleukin 4 and interleukin 5 lead to containment of different worm . CC-BY 4.0 International license made available under a (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.11.499516 doi: bioRxiv preprint material, further inquiries can be directed to the corresponding author/s.  (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is The copyright holder for this preprint this version posted July 12, 2022. ; https://doi.org/10.1101/2022.07.11.499516 doi: bioRxiv preprint