scholarly journals Avirulent Uracil Auxotrophs Based on Disruption of Orotidine-5′-Monophosphate Decarboxylase Elicit Protective Immunity to Toxoplasma gondii

2010 ◽  
Vol 78 (9) ◽  
pp. 3744-3752 ◽  
Author(s):  
Barbara A. Fox ◽  
David J. Bzik
Keyword(s):  
Toxoplasma Gondii ◽  
Protective Immunity ◽  
De Novo ◽  
Genetically Engineered ◽  
Type I ◽  
Double Knockout ◽  
Lethal Challenge ◽  
Knockout Strain ◽  
Monophosphate Decarboxylase ◽  
Uracil Auxotrophs

ABSTRACT The orotidine-5′-monophosphate decarboxylase (OMPDC) gene, encoding the final enzyme of the de novo pyrimidine biosynthesis pathway, was deleted using Toxoplasma gondii KU80 knockouts to develop an avirulent nonreverting pyrimidine auxotroph strain. Additionally, to functionally address the role of the pyrimidine salvage pathway, the uridine phosphorylase (UP) salvage activity was knocked out and a double knockout of UP and OMPDC was also constructed. The nonreverting ΔOMPDC, ΔUP, and ΔOMPDC ΔUP knockout strains were evaluated for pyrimidine auxotrophy, for attenuation of virulence, and for their ability to elicit potent immunity to reinfection. The ΔUP knockout strain was replication competent and virulent. In contrast, the ΔOMPDC and ΔOMPDC ΔUP strains were uracil auxotrophs that rapidly lost their viability during pyrimidine starvation. Replication of the ΔOMPDC strain but not the ΔOMPDC ΔUP strain was also partially rescued in vitro with uridine or cytidine supplementation. Compared to their hypervirulent parental type I strain, the ΔOMPDC and ΔOMPDC ΔUP knockout strains exhibited extreme attenuation in murine virulence (∼8 logs). Genetic complementation of the ΔOMPDC strain using a functional OMPDC allele restored normal replication and type I parental strain virulence phenotypes. A single immunization of mice with either the live critically attenuated ΔOMPDC strain or the ΔOMPDC ΔUP knockout strain effectively induced potent protective immunity to lethal challenge infection. The avirulent nonreverting ΔOMPDC and ΔOMPDC ΔUP strains provide new tools for the dissection of the host response to infection and are promising candidates for safe and effective Th1 vaccine platforms that can be easily genetically engineered.

scholarly journals Live Chimeric and Inactivated Japanese Encephalitis Virus Vaccines Differ in Their Cross-Protective Values against Murray Valley Encephalitis Virus

2008 ◽  
Vol 83 (6) ◽  
pp. 2436-2445 ◽  
Author(s):  
Mario Lobigs ◽  
Maximilian Larena ◽  
Mohammed Alsharifi ◽  
Eva Lee ◽  
Megan Pavy
Keyword(s):  
Japanese Encephalitis Virus ◽  
Japanese Encephalitis ◽  
Protective Immunity ◽  
Encephalitis Virus ◽  
Type I ◽  
Murray Valley Encephalitis Virus ◽  
Lethal Challenge ◽  
Live Virus ◽  
Immunodeficient Mice ◽  
The Cross

ABSTRACT The Japanese encephalitis virus (JEV) serocomplex, which also includes Murray Valley encephalitis virus (MVEV), is a group of antigenically closely related, mosquito-borne flaviviruses that are responsible for severe encephalitic disease in humans. While vaccines against the prominent members of this serocomplex are available or under development, it is unlikely that they will be produced specifically against those viruses which cause less-frequent disease, such as MVEV. Here we have evaluated the cross-protective values of an inactivated JEV vaccine (JE-VAX) and a live chimeric JEV vaccine (ChimeriVax-JE) against MVEV in two mouse models of flaviviral encephalitis. We show that (i) a three-dose vaccination schedule with JE-VAX provides cross-protective immunity, albeit only partial in the more severe challenge model; (ii) a single dose of ChimeriVax-JE gives complete protection in both challenge models; (iii) the cross-protective immunity elicited with ChimeriVax-JE is durable (≥5 months) and broad (also giving protection against West Nile virus); (iv) humoral and cellular immunities elicited with ChimeriVax-JE contribute to protection against lethal challenge with MVEV; (v) ChimeriVax-JE remains fully attenuated in immunodeficient mice lacking type I and type II interferon responses; and (vi) immunization with JE-VAX, but not ChimeriVax-JE, can prime heterologous infection enhancement in recipients of vaccination on a low-dose schedule, designed to mimic vaccine failure or waning of vaccine-induced immunity. Our results suggest that the live chimeric JEV vaccine will protect against other viruses belonging to the JEV serocomplex, consistent with the observation of cross-protection following live virus infections.

scholarly journals Targeted Disruption of the Inosine 5′-Monophosphate Dehydrogenase Type I Gene in Mice

2003 ◽  
Vol 23 (18) ◽  
pp. 6702-6712 ◽  
Author(s):  
Jing Jin Gu ◽  
Amy K. Tolin ◽  
Jugnu Jain ◽  
Hai Huang ◽  
Lalaine Santiago ◽  
...  
Keyword(s):  
T Cell Activation ◽  
Cell Activation ◽  
De Novo ◽  
Cytolytic Activity ◽  
Guanine Nucleotides ◽  
Interleukin 4 ◽  
Type I ◽  
Double Knockout ◽  
Inhibition Of Proliferation ◽  
Monophosphate Dehydrogenase

ABSTRACT Inosine 5′-monophosphate dehydrogenase (IMPDH) is the critical, rate-limiting enzyme in the de novo biosynthesis pathway for guanine nucleotides. Two separate isoenzymes, designated IMPDH types I and II, contribute to IMPDH activity. An additional pathway salvages guanine through the activity of hypoxanthine-guanine phosphoribosyltransferase (HPRT) to supply the cell with guanine nucleotides. In order to better understand the relative contributions of IMPDH types I and II and HPRT to normal biological function, a mouse deficient in IMPDH type I was generated by standard gene-targeting techniques and bred to mice deficient in HPRT or heterozygous for IMPDH type II. T-cell activation in response to anti-CD3 plus anti-CD28 antibodies was significantly impaired in both single- and double-knockout mice, whereas a more general inhibition of proliferation in response to other T- and B-cell mitogens was observed only in mice deficient in both enzymes. In addition, IMPDH type I−/− HPRT−/0 splenocytes showed reduced interleukin-4 production and impaired cytolytic activity after antibody activation, indicating an important role for guanine salvage in supplementing the de novo synthesis of guanine nucleotides. We conclude that both IMPDH and HPRT activities contribute to normal T-lymphocyte activation and function.

A single dose of ChAdOx1 MERS provides broad protective immunity against a variety of MERS-CoV strains

2020 ◽  
Author(s):  
Neeltje van Doremalen ◽  
Elaine Haddock ◽  
Friederike Feldmann ◽  
Kimberly Meade-White ◽  
Trenton Bushmaker ◽  
...  
Keyword(s):  
Single Dose ◽  
Middle East ◽  
Protective Immunity ◽  
Rhesus Macaques ◽  
Clinical Development ◽  
Dromedary Camel ◽  
Type I ◽  
Dromedary Camels ◽  
Lethal Challenge ◽  
Antibody Titers

AbstractMiddle East respiratory syndrome coronavirus (MERS-CoV) continues to infect humans via the dromedary camel reservoir and can transmit between humans, most commonly via nosocomial transmission. Currently, no licensed vaccine is available. Previously we showed that vaccination of transgenic mice with ChAdOx1 MERS, encoding the MERS S protein, prevented disease upon lethal challenge. In the current study we show that rhesus macaques seroconverted rapidly after a single intramuscular vaccination with ChAdOx1 MERS. Upon MERS-CoV challenge vaccinated animals were protected against respiratory injury and pneumonia and had a reduction in viral load in lung tissue of several logs. Furthermore, we did not detect MERS-CoV replication in type I and II pneumocytes of ChAdOx1 MERS vaccinated animals. A prime-boost regimen of ChAdOx1 MERS boosted antibody titers, and viral replication was completely absent from the respiratory tract tissue of these rhesus macaques. Finally, we investigated the ability of ChAdOx1 MERS to protect against six different MERS-CoV strains, isolated between 2012 to 2018, from dromedary camels and humans in the Middle East and Africa. Antibodies elicited by ChAdOx1 MERS in rhesus macaques were able to neutralize all MERS-CoV strains. Vaccination of transgenic hDPP4 mice with ChAdOx1 MERS completely protected the animals against disease and lethality for all different MERS-CoV strains. The data support further clinical development of ChAdOx1 MERS supported by CEPI.One Sentence SummaryPrime-only vaccination with ChAdOx1 MERS provides protective immunity against HCoV-EMC/2012 replication in rhesus macaques, and a wide variety of MERS-CoV strains in mice.

scholarly journals Nonreplicating, Cyst-Defective Type II Toxoplasma gondii Vaccine Strains Stimulate Protective Immunity against Acute and Chronic Infection

2015 ◽  
Vol 83 (5) ◽  
pp. 2148-2155 ◽  
Author(s):  
Barbara A. Fox ◽  
David J. Bzik
Keyword(s):  
Toxoplasma Gondii ◽  
Chronic Infection ◽  
Acute Infection ◽  
Cyst Formation ◽  
Type I ◽  
Type Ii ◽  
Live Attenuated Vaccine ◽  
Content Type ◽  
Monophosphate Decarboxylase ◽  
Uracil Auxotroph

Live attenuated vaccine strains, such as type I nonreplicating uracil auxotroph mutants, are highly effective in eliciting lifelong immunity to virulent acute infection byToxoplasma gondii. However, it is currently unknown whether vaccine-elicited immunity can provide protection against acute infection and also prevent chronic infection. To address this problem, we developed nonreverting, nonreplicating, live attenuated uracil auxotroph vaccine strains in the type II Δku80genetic background by targeting the deletion of the orotidine 5′-monophosphate decarboxylase (OMPDC) and uridine phosphorylase (UP) genes. Deletion ofOMPDCinduced a severe uracil auxotrophy with loss of replication, loss of virulence in mice, and loss of the ability to develop cysts and chronic infection. Vaccination of mice using type II Δku80Δompdcmutants stimulated a fully protective CD8+T cell-dependent immunity that prevented acute infection by type I and type II strains ofT. gondii, and this vaccination also severely reduced or prevented cyst formation after type II challenge infection. Nonreverting, nonreplicating, and non-cyst-forming Δompdcmutants provide new tools to examine protective immune responses elicited by vaccination with a live attenuated type II vaccine.

scholarly journals AMA1-Deficient Toxoplasma gondii Parasites Transiently Colonize Mice and Trigger an Innate Immune Response That Leads to Long-Lasting Protective Immunity

2015 ◽  
Vol 83 (6) ◽  
pp. 2475-2486 ◽  
Author(s):  
Vanessa Lagal ◽  
Márcia Dinis ◽  
Dominique Cannella ◽  
Daniel Bargieri ◽  
Virginie Gonzalez ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Distinct Type ◽  
Genetically Engineered ◽  
Host Cells ◽  
Type I ◽  
Content Type ◽  
Apical Membrane Antigen 1 ◽  
Immunocompromised Mice

The apical membrane antigen 1 (AMA1) protein was believed to be essential for the perpetuation of two Apicomplexa parasite genera,PlasmodiumandToxoplasma, until we genetically engineered viable parasites lackingAMA1. The reduction in invasiveness of theToxoplasma gondiiRH-AMA1 knockout (RH-AMA1KO) tachyzoite population,in vitro, raised key questions about the outcome associated with these tachyzoites once inoculated in the peritoneal cavity of mice. In this study, we used AMNIS technology to simultaneously quantify and image the parasitic process driven by AMA1KOtachyzoites. We report their ability to colonize and multiply in mesothelial cells and in both resident and recruited leukocytes. While the RH-AMA1KOpopulation amplification is rapidly lethal in immunocompromised mice, it is controlled in immunocompetent hosts, where immune cells in combination sense parasites and secrete proinflammatory cytokines. This innate response further leads to a long-lasting status immunoprotective against a secondary challenge by high inocula of the homologous type I or a distinct type IIT. gondiigenotypes. While AMA1 is definitively not an essential protein for tachyzoite entry and multiplication in host cells, it clearly assists the expansion of parasite populationin vivo.

scholarly journals Requirement of Toxoplasma gondii metacaspases for IMC1 maturation, endodyogeny and virulence in mice

2021 ◽  
Vol 14 (1) ◽  
Author(s):  
Muzi Li ◽  
Jing Liu ◽  
Yayun Wu ◽  
Yihan Wu ◽  
Xiaodong Sun ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Normal Growth ◽  
Double Knockout ◽  
Protein Aggregate ◽  
Knockout Strain ◽  
Membrane Complex ◽  
Inner Membrane Complex ◽  
Single Knockout

Abstract Background Metacaspases are multifunctional proteins found in plants, fungi and protozoa, and are involved in processes such as insoluble protein aggregate clearance and cell proliferation. Our previous study demonstrated that metacaspase-1 (MCA1) contributes to parasite apoptosis in Toxoplasma gondii. Deletion of MCA1 from T. gondii has no effect on the growth and virulence of the parasites. Three metacaspases were identified in the ToxoDB Toxoplasma Informatics Resource, and the function of metacaspase-2 (MCA2) and metacaspase-3 (MCA3) has not been demonstrated. Methods In this study, we constructed MCA1, MCA2 and MCA1/MCA2 transgenic strains from RHΔku80 (Δku80), including overexpressing strains and knockout strains, to clarify the function of MCA1 and MCA2 of T. gondii. Results MCA1 and MCA2 were distributed in the cytoplasm with punctuated aggregation, and part of the punctuated aggregation of MCA1 and MCA2 was localized on the inner membrane complex of T. gondii. The proliferation of the MCA1/MCA2 double-knockout strain was significantly reduced; however, the two single knockout strains (MCA1 knockout strain and MCA2 knockout strain) exhibited normal growth rates as compared to the parental strain, Δku80. In addition, endodyogeny was impaired in the tachyzoites whose MCA1 and MCA2 were both deleted due to multiple nuclei and abnormal expression of IMC1. We further found that IMC1 of the double-knockout strain was detergent-soluble, indicating that MCA1 and MCA2 are associated with IMC1 maturation. Compared to the parental Δku80 strain, the double-knockout strain was more readily induced from tachyzoites to bradyzoites in vitro. Furthermore, the double-knockout strain was less pathogenic in mice and was able to develop bradyzoites in the brain, which formed cysts and established chronic infection. Conclusion MCA1 and MCA2 are important factors which participate in IMC1 maturation and endodyogeny of T. gondii. The double-knockout strain has slower proliferation and was able to develop bradyzoites both in vitro and in vivo. Graphic abstract

scholarly journals Long-Term Protective Immune Response Elicited by Vaccination with an Expression Genomic Library of Toxoplasma gondii

2003 ◽  
Vol 71 (9) ◽  
pp. 5407-5411 ◽  
Author(s):  
Alberto Fachado ◽  
Alexandro Rodriguez ◽  
Judith Molina ◽  
Jaline C. Silvério ◽  
Ana P. M. P. Marino ◽  
...  
Keyword(s):  
Immune Response ◽  
Toxoplasma Gondii ◽  
Dna Vaccine ◽  
Protective Immunity ◽  
Genomic Library ◽  
Protective Immune Response ◽  
Lethal Challenge

ABSTRACT Immunization of BALB/c mice with an expression genomic library of Toxoplasma gondii induces a Th1-type immune response, with recognition of several T. gondii proteins (21 to 117 kDa) and long-term protective immunity against a lethal challenge. These results support further investigations to achieve a multicomponent anti-T. gondii DNA vaccine.

scholarly journals Apicoplast isoprenoid precursor synthesis and the molecular basis of fosmidomycin resistance in Toxoplasma gondii

2011 ◽  
Vol 208 (7) ◽  
pp. 1547-1559 ◽  
Author(s):  
Sethu C. Nair ◽  
Carrie F. Brooks ◽  
Christopher D. Goodman ◽  
Angelika Strurm ◽  
Geoffrey I. McFadden ◽  
...  
Keyword(s):  
Toxoplasma Gondii ◽  
Molecular Basis ◽  
De Novo ◽  
Strong Support ◽  
Specific Inhibitor ◽  
Drug Susceptibility ◽  
Drug Uptake ◽  
Lethal Challenge ◽  
Transporter Protein ◽  
Metabolite Exchange

Apicomplexa are important pathogens that include the causative agents of malaria, toxoplasmosis, and cryptosporidiosis. Apicomplexan parasites contain a relict chloroplast, the apicoplast. The apicoplast is indispensable and an attractive drug target. The apicoplast is home to a 1-deoxy-d-xylulose-5-phosphate (DOXP) pathway for the synthesis of isoprenoid precursors. This pathway is believed to be the most conserved function of the apicoplast, and fosmidomycin, a specific inhibitor of the pathway, is an effective antimalarial. Surprisingly, fosmidomycin has no effect on most other apicomplexans. Using Toxoplasma gondii, we establish that the pathway is essential in parasites that are highly fosmidomycin resistant. We define the molecular basis of resistance and susceptibility, experimentally testing various host and parasite contributions in T. gondii and Plasmodium. We demonstrate that in T. gondii the parasite plasma membrane is a critical barrier to drug uptake. In strong support of this hypothesis, we engineer de novo drug-sensitive T. gondii parasites by heterologous expression of a bacterial transporter protein. Mice infected with these transgenic parasites can now be cured from a lethal challenge with fosmidomycin. We propose that the varied extent of metabolite exchange between host and parasite is a crucial determinator of drug susceptibility and a predictor of future resistance.

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