Di-cationic arylimidamides Act Against Neospora caninum tachyzoites by Interference in Membrane Structure and Nucleolar Integrity and are Active Against Challenge Infection in Mice

Neospora caninum is considered to be the main cause of bovine abortion in Europe and the USA, leading to considerable financial impact. Losses are caused directly by abortions or indirectly through breeding of calves with impaired viability. Due to the lack of effective chemotherapy against bovine neosporosis, there is a need to develop new anti-protozoal compounds, which would either eliminate the parasite or avoid its transmission. In order to identify compounds of interest, the in vitro activities of 41 di-cationic pentamidine derivatives were studied employing a transgenic N. caninum clone expressing beta-galactosidase as a reporter gene. The arylimidamide DB745, previously shown to be highly active against Leishmania donovani in vitro and in vivo, appeared as the most promising compound, with an IC50 of 80 nM in 3-day growth assays and severely affecting both host cell invasion as well as intracellular proliferation. TEM of intracellular tachyzoites identified distinct alterations related to the nucleolus and the nuclear and cellular membrane. Long-term growth assays showed that DB745 acted parasiticidal upon the Nc-Liv isolate, but not against the Nc-1 isolate of N. caninum. In vivo studies in N. caninum (Nc-1 isolate) infected mice showed that daily intraperitoneal application of DB745 for a period of 14 days resulted in a decreased number of clinically affected animals, and lower cerebral parasite burdens in DB745-treated mice compared to non-treated mice. These results illustrate the potential of dicationic arylimidamides for the treatment of N. caninum infections

Use of a Th1 Stimulator Adjuvant for Vaccination against Neospora caninum Infection in the Pregnant Mouse Model

Vertical transmission from an infected cow to its fetus accounts for the vast majority of new Neospora caninum infections in cattle. A vaccine composed of a chimeric antigen named recNcMIC3-1-R, based on predicted immunogenic domains of the two microneme proteins NcMIC1 and NcMIC3, the rhoptry protein NcROP2, and emulsified in saponin adjuvants, significantly reduced the cerebral infection in non-pregnant BALB/c mice. Protection was associated with a mixed Th1/Th2-type cytokine response. However, the same vaccine formulation elicited a Th2-type immune response in pregnant mice and did not prevent vertical transmission or disease, neither in dams nor in offspring mice. In this study, an alternative vaccine formulation containing recNcMIC3-1-R emulsified in Freund’s incomplete adjuvant, a stimulator of the cellular immunity, was investigated. No protection against vertical transmission and cerebral infection in the pregnant mice and a very limited protective effect in the non-pregnant mice were observed. The vaccine induced a Th1-type immune response characterized by high IgG2a titres and strong IFN-γ expression, which appeared detrimental to pregnancy

Vaccines against neosporosis: what can we learn from the past studies?

Neospora caninum is an intracellular apicomplexan parasite, which is a leading cause of abortion in cattle; thus neosporosis represents an important veterinary health problem and is of high economic significance. The parasite can infect cattle via trans-placental transmission from an infected cow to its fetus (vertical transmission), or through the oral route via ingestion of food or water contaminated with oocysts that were previously shed with the feces of a canid definitive host (horizontal transmission). Although vaccination was considered a rational strategy to prevent bovine neosporosis, the only commercialized vaccine (Neoguard®) produced ambiguous results with relatively low efficacy, and was recently removed from the market. Therefore, there is a need to develop an efficient vaccine capable of preventing both, the horizontal transmission through infected food or water to a naïve animal as well as the vertical transmission from infected but clinically asymptomatic dams to the fetus. Different vaccine strategies have been investigated, including the use of live attenuated vaccines, killed parasite lysates, total antigens or antigen fractions from killed parasites, and subunit vaccines. The vast majority of experimental studies were performed in mice, and to a certain extent in gerbils, but there is also a large number of investigations that were conducted in cattle and sheep. However, it is difficult to directly compare these studies due to the high variability of the parameters employed. In this review, we will summarize the recent advances made in vaccine development against N. caninum in cattle and in mice and highlight the most important factors, which are likely to influence the degree of protection mediated by vaccination

Immunoglobulin domain interface exchange as a platform technology for the generation of Fc heterodimers and bispecific antibodies

Bispecific antibodies (bsAbs) are of significant importance to the development of novel antibody-based therapies, and heavy chain (Hc) heterodimers represent a major class of bispecific drug candidates. Current technologies for the generation of Hc heterodimers are suboptimal and often suffer from contamination by homodimers posing purification challenges. Here, we introduce a new technology based on biomimicry wherein the protein-protein interfaces of two different immunoglobulin (Ig) constant domain pairs are exchanged in part or fully to design new heterodimeric domains. The method can be applied across Igs to design Fc heterodimers and bsAbs. We investigated interfaces from human IgA CH3, IgD CH3, IgG1 CH3, IgM CH4, T-cell receptor (TCR) α/β, and TCR γ/δ constant domain pairs, and we found that they successfully drive human IgG1 CH3 or IgM CH4 heterodimerization to levels similar to or above those of reference methods. A comprehensive interface exchange between the TCR α/β constant domain pair and the IgG1 CH3 homodimer was evidenced by X-ray crystallography and used to engineer examples of bsAbs for cancer therapy. Parental antibody pairs were rapidly reformatted into scalable bsAbs that were free of homodimer traces by combining interface exchange, asymmetric Protein A binding, and the scFv × Fab format. In summary, we successfully built several new CH3- or CH4-based heterodimers that may prove useful for designing new bsAb-based therapeutics, and we anticipate that our approach could be broadly implemented across the Ig constant domain family. To our knowledge, CH4-based heterodimers have not been previously reported

Differential Effects on Survival, Humoral Immune Responses and Brain Lesions in Inbred BALB/C, CBA/CA, and C57BL/6 Mice Experimentally Infected with Neospora caninum Tachyzoites

C57BL/6, BALB/c, and CBA/Ca mouse strains with different MHC-I haplotypes were compared with respect to susceptibility to Neospora caninum infection. Groups of 5 mice received , , or tachyzoites of the NC-Liverpool isolate by intraperitoneal injection and were observed for disease symptoms. Humoral responses, splenocyte interferon-γ (IFN-γ) production, cerebral parasite loads, and histopathology were evaluated at human end points or the latest at 34 days postinfection (PI). The mortality rates in C57BL/6 mice were the highest, and relatively high levels of IgG1 antibodies were detected in those mice surviving till 34 days PI. In lymphocyte proliferation assays, spleen cells from C57BL6 mice stimulated with N. caninum antigen extract exhibited large variations in IFN-γ production. In BALB/c mice mortality was 0% at the lowest and 100% at the highest infection dose. Serologically they responded with high levels of both IgG2a and IgG1 subclasses, and lymphocyte proliferation assays of surviving mice yielded lower IFN-γ levels. CBA/Ca mice were the most resistant, with no animal succumbing to infection at a dose of and tachyzoites, but 100% mortality at tachyzoites. High IgG2a levels as well as increased IFN-γ in lymphocyte proliferation assays were measured in CBA/Ca mice infected with tachyzoites

Vaccination with the recombinant chimeric antigen recNcMIC3-1-R induces a non-protective Th2-type immune response in the pregnant mouse model for N. caninum infection

The major route of transmission of Neospora caninum in cattle is transplacentally from an infected cow to its progeny. Therefore, a vaccine should be able to prevent both the horizontal transmission from contaminated food or water and the vertical transmission. We have previously shown that a chimeric vaccine composed of predicted immunogenic epitopes of NcMIC3, NcMIC1 and NcROP2 (recNcMIC3-1-R) significantly reduced the cerebral infection in BALB/c mice. In this study, mice were first vaccinated, then mated and pregnant mice were challenged with 2×10(6)N. caninum tachyzoites at day 7-9 of pregnancy. Partial protection was only observed in the mice vaccinated with a tachyzoite crude protein extract but no protection against vertical transmission or cerebral infection in the dams was observed in the group vaccinated with recNcMIC3-1-R. Serological and cytokine analysis showed an overall lower cytokine level in sera associated with a dominant IL-4 expression and high IgG1 titers. Thus, the Th2-type immune response observed in the pregnant mice was not protective against experimental neosporosis, in contrary to the mixed Th1-/Th2-type immune response observed in the non-pregnant mouse model. These results demonstrate that the immunomodulation that occurs during pregnancy was not favorable for the protection against N. caninum infection conferred by vaccination with recNcMIC3-1-R

Can we use isotopic analysis on archaeological charcoals as paleoenvironmental proxy for Pleistocene sequences?

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RecNcMIC3-1-R is a microneme- and rhoptry-based chimeric antigen that protects against acute neosporosis and limits cerebral parasite load in the mouse model for Neospora caninum infection

In order to achieve host cell entry, the apicomplexan parasite Neospora caninum relies on the contents of distinct organelles, named micronemes, rhoptries and dense granules, which are secreted at defined timepoints during and after host cell entry. It was shown previously that a vaccine composed of a mixture of three recombinant antigens, corresponding to the two microneme antigens NcMIC1 and NcMIC3 and the rhoptry protein NcROP2, prevented disease and limited cerebral infection and transplacental transmission in mice. In this study, we selected predicted immunogenic domains of each of these proteins and created four different chimeric antigens, with the respective domains incorporated into these chimers in different orders. Following vaccination, mice were challenged intraperitoneally with 2 × 10(6)N. caninum tachzyoites and were then carefully monitored for clinical symptoms during 4 weeks post-infection. Of the four chimeric antigens, only recNcMIC3-1-R provided complete protection against disease with 100% survivors, compared to 40-80% of survivors in the other groups. Serology did not show any clear differences in total IgG, IgG1 and IgG2a levels between the different treatment groups. Vaccination with all four chimeric variants generated an IL-4 biased cytokine expression, which then shifted to an IFN-γ-dominated response following experimental infection. Sera of recNcMIC3-1-R vaccinated mice reacted with each individual recombinant antigen, as well as with three distinct bands in Neospora extracts with similar Mr as NcMIC1, NcMIC3 and NcROP2, and exhibited distinct apical labeling in tachyzoites. These results suggest that recNcMIC3-1-R is an interesting chimeric vaccine candidate and should be followed up in subsequent studies in a fetal infection model