Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs

Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV–LACK, rCDV–TSA, and rCDV–LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV–LACK showed markedly smaller nodules without ulceration. Although the rCDV–TSA- and rCDV–LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV–LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs

Efficacy of Recombinant Canine Distemper Virus Expressing Leishmania Antigen against Leishmania Challenge in Dogs.

Canine distemper virus (CDV) vaccination confers long-term protection against CDV reinfection. To investigate the utility of CDV as a polyvalent vaccine vector for Leishmania, we generated recombinant CDVs, based on an avirulent Yanaka strain, that expressed Leishmania antigens: LACK, TSA, or LmSTI1 (rCDV-LACK, rCDV-TSA, and rCDV-LmSTI1, respectively). Dogs immunized with rCDV-LACK were protected against challenge with lethal doses of virulent CDV, in the same way as the parental Yanaka strain. To evaluate the protective effects of the recombinant CDVs against cutaneous leishmaniasis in dogs, dogs were immunized with one recombinant CDV or a cocktail of three recombinant CDVs, before intradermal challenge (in the ears) with infective-stage promastigotes of Leishmania major. Unvaccinated dogs showed increased nodules with ulcer formation after 3 weeks, whereas dogs immunized with rCDV-LACK showed markedly smaller nodules without ulceration. Although the rCDV-TSA- and rCDV-LmSTI1-immunized dogs showed little protection against L. major, the cocktail of three recombinant CDVs more effectively suppressed the progression of nodule formation than immunization with rCDV-LACK alone. These results indicate that recombinant CDV is suitable for use as a polyvalent live attenuated vaccine for protection against both CDV and L. major infections in dogs

Changes in body temperatures and leukocyte counts after virulent CDV challenge.

<p>The body temperatures and leucocyte counts of mock- and rCDV-LACK-immunized dogs after challenge with the virulent Snyder Hill strain of CDV were measured daily. At 7 days after challenge, the mock-treated dogs were in a moribund state and were euthanized (†). Each symbol represents one animal.</p

Generation and <i>in vitro</i> characterization of rCDVs.

<p>(A) Schematic model of pCDV (Yanaka) with the <i>Fse</i>I site introduced between the N and P genes. Each <i>Leishmania</i> antigen gene carrying the CDV transcription signal unit (lower case) and <i>Fse</i>I site (underlined) was inserted at the <i>Fse</i>I site. (B) The rescued recombinant viruses were identified by immunoblotting analysis. Cell lysates were examined with anti-CDV-N, anti-LACK, anti-TSA and anti-LmSTI1 antibodies. The bands corresponding to CDV-N (68 kDa), LACK (38 kDa), TSA (22 kDa) and LmSTI1 (62 kDa) are indicated by arrowheads. (C) Kinetics of recombinant viral growth. B95a cells were inoculated with a recombinant virus at a multiplicity of infection of 0.01, and then harvested on the indicated day. The titers of the viruses were determined using a TCID₅₀ assay.</p

Time course of skin lesion development in dogs infected with <i>L</i>. <i>major</i>.

<p>(A) Beagle dogs were infected intradermally (in the ears) with 5 × 10⁷ infective promastigotes of <i>L</i>. <i>major</i> per spot, and the lesion sizes were measured weekly. Parasite growth was evaluated as nodule size. Three independent spots per dog were determined and followed-up. Data are shown as means ± SEM, and the error bars reflect the three inoculated spots. (B) Images of lesions at 2 to 5 weeks after infection.</p

Protective efficacy of immunization with rCDV-LACK, rCDV-TSA and rCDV-LmSTI1 against <i>L</i>. <i>major</i> challenge.

<p>(A) Dogs were inoculated intradermally (in the ears) with 5 × 10⁷ infective promastigotes of <i>L</i>. <i>major</i> per spot, 42 days after their first vaccination with the indicated inoculum. Parasite growth was evaluated every week as nodule size. Three independent spots were inoculated per dog and followed-up. Data are shown as the mean ± SEM, and the error bars reflect the three inoculated spots. (B) Photographs of the nodules on the ears of mock- and rCDV-LACK-immunized dogs in the fourth week after <i>L</i>. <i>major</i> challenge.</p

NPM1-mutation-based measurable residual disease assessment after completion of two courses of post-remission therapy is a valuable clinical predictor of the prognosis of acute myeloid leukemia

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