Vaccination using live attenuated Leishmania donovani centrin deleted parasites induces protection in dogs against Leishmania infantum

Live attenuated Leishmania donovani parasites such as LdCen^−/− have been shown elicit protective immunity against leishmanial infection in mice and hamster models. Previously, we have reported on the induction of strong immunogenicity in dogs upon vaccination with LdCen^−/− including an increase in immunoglobulin isotypes, higher lymphoproliferative response, higher frequencies of activated CD4⁺ and CD8⁺ T cells, IFN-γ production by CD8⁺ T cells, increased secretion of TNF-α and IL-12/IL-23p40 and, finally, decreased secretion of IL-4. To further explore the potential of LdCen^−/− parasites as vaccine candidates, we performed a 24-month follow up of LdCen^−/− immunized dogs after challenge with virulent Leishmania infantum, aiming determination of parasite burden by qPCR, antibody production (ELISA) and cellular responses (T cell activation and cytokine production) by flow cytometry and sandwich ELISA. Our data demonstrated that vaccination with a single dose of LdCen^−/− (without any adjuvant) resulted in the reduction of up to 87.3% of parasite burden after 18 months of virulent challenge. These results are comparable to those obtained with commercially available vaccine in Brazil (Leishmune^®). The protection was associated with antibody production and CD4⁺ and CD8⁺ proliferative responses, as well as T cell activation and significantly higher production of IFN-γ, IL-12/IL-23p40 and TNF-α, which was comparable to responses induced by immunization with Leishmune^®, with significant differences when compared to control animals (Placebo). Moreover, only animals immunized with LdCen^−/− expressed lower levels of IL-4 when compared to animals vaccinated either with Leishmune^® or PBS. Our results support further studies aiming to demonstrate the potential of genetically modified live attenuated L. donovani vaccine to control L. infantum transmission in endemic areas for CVL.     

The Chimerical Multi-Component Q protein from Leishmania in the absence of adjuvant protects dogs against an experimental Leishmania infantum infection

The protective potential against Leishmania infection of the Leishmania chimerical Q protein administered as a single (Q) or double dose (Q+Q) without adjuvant was analyzed in a double-blind placebo controlled experiment in dogs. During vaccination the protein induced an intense early anti-Q response but no reactivity against total Leishmania infantum proteins was detected. Several end-points were taken into consideration. In the vaccinated animals the amount and intensity of clinical symptoms was lower than in the control group. Pathological signs of disease were observed in liver, kidney and spleen of all dogs from the control group in contrast to the normal appearance of the organs of the vaccinated animals. Vaccination was able to induce parasite clearance in most dogs. Only 1/7 dog was parasite DNA positive in skin in the Q group in contrast to 6/7 dogs in control and 4/7 in Q+Q. Significant anti-SLA clearance was observed in the vaccinated animals at the end of the study. Differences between control and vaccinated animals were also observed at the biochemical level, DTH and nitrite oxide production.     

Genome-wide SNP and microsatellite variation illuminate population-level epidemiology in the Leishmania donovani species complex

The species of the Leishmania donovani species complex cause visceral leishmaniasis, a debilitating infectious disease transmitted by sandflies. Understanding molecular changes associated with population structure in these parasites can help unravel their epidemiology and spread in humans. In this study, we used a panel of standard microsatellite loci and genome-wide SNPs to investigate population-level diversity in L. donovani strains recently isolated from a small geographic area spanning India, Bihar and Nepal, and compared their variation to that found in diverse strains of the L. donovani complex isolates from Europe, Africa and Asia. Microsatellites and SNPs could clearly resolve the phylogenetic relationships of the strains between continents, and microsatellite phylogenies indicated that certain older Indian strains were closely related to African strains. In the context of the anti-malaria spraying campaigns in the 1960s, this was consistent with a pattern of episodic population size contractions and clonal expansions in these parasites that was supported by population history simulations. In sharp contrast to the low resolution provided by microsatellites, SNPs retained a much more fine-scale resolution of population-level variability to the extent that they identified four different lineages from the same region one of which was more closely related to African and European strains than to Indian or Nepalese ones. Joining results of in vitro testing the antimonial drug sensitivity with the phylogenetic signals from the SNP data highlighted protein-level mutations revealing a distinct drug-resistant group of Nepalese and Indian L. donovani. This study demonstrates the power of genomic data for exploring parasite population structure. Furthermore, markers defining different genetic groups have been discovered that could potentially be applied to investigate drug resistance in clinical Leishmania strains.     

Leishmanization revisited: Immunization with a naturally attenuated cutaneous Leishmania donovani isolate from Sri Lanka protects against visceral leishmaniasis

Leishmaniasis is a neglected tropical disease caused by Leishmania protozoa and associated with three main clinical presentations: cutaneous, mucocutaneous and visceral leishmaniasis. Visceral leishmaniasis is the second most lethal parasitic disease after malaria and there is so far no human vaccine. Leishmania donovani is a causative agent of visceral leishmaniasis in South East Asia and Eastern Africa. However, in Sri Lanka, L. donovani causes mainly cutaneous leishmaniasis, while visceral leishmaniasis is rare. We investigate here the possibility that the cutaneous form of L. donovani can provide immunological protection against the visceral form of the disease, as a potential explanation for why visceral leishmaniasis is rare in Sri Lanka. Subcutaneous immunization with a cutaneous clinical isolate from Sri Lanka was significantly protective against visceral leishmaniasis in BALB/c mice. Protection was associated with a mixed Th1/Th2 response. These results provide a possible rationale for the scarcity of visceral leishmaniasis in Sri Lanka and could guide leishmaniasis vaccine development efforts.     

Safety and immunogenicity of live,attenuated intranasal Bordetella pertussis vaccine (BPZE1) in healthy adults

BackgroundBPZE1 is a live, attenuated pertussis vaccine derived from B. pertussis strain Tohama I modified by genetic removal or inactivation of 3 B. pertussis toxins: pertussis toxin, dermonecrotic toxin, and tracheal cytotoxin. This Phase 2a study evaluated the safety and immunogenicity of liquid or lyophilized BPZE1 vaccine administered intranasally by needleless tuberculin syringe or mucosal atomization device (VaxINator^TM) at two dose levels.MethodsFifty healthy male and non-pregnant female participants 18–49 years of age were enrolled. Participants were randomized 3:3:3:1 to a single lyophilized dose of 10⁷ colony forming units (CFU) BPZE1, 10⁹ CFU BPZE1, placebo via VaxINator device, or a single liquid dose of 10⁹ CFU BPZE1 via tuberculin syringe. Reactogenicity was assessed for 14 days. Blood was obtained pre-vaccination; on Day 8 (safety); and on Days 15, 29, and 181 (immunogenicity). Nasal wick and swab samples were obtained at baseline and on Days 29 and 181 for assessment of mucosal antibody responses and clearance of BPZE1.ResultsAcross all groups, 35/50 (70 %) experienced at least one local adverse event (AE) and 31/50 (62 %) experienced at least one systemic AE, with similar AE frequencies observed between the highest 10⁹ CFU BPZE1 and placebo groups. There were no severe or serious AEs during the study. At Day 29, seroconversion (≥2-fold rise from baseline in serum IgG or IgA) to at least 2 pertussis antigens was observed in 73 % in the 10⁹ CFU BPZE1 VaxINator group, 60 % in the 10⁹ CFU BPZE1 group delivered via tuberculin syringe, 27 % of participants in the 10⁷ CFU BPZE1 VaxINator group, and 20 % in the placebo VaxINator group. No participants were colonized with BPZE1 at Day 29 post vaccination.DiscussionLyophilized BPZE1 vaccine was well tolerated and immunogenic at the highest dose (10⁹ CFU) delivered intranasally by VaxINator device and was not associated with any SAEs or prolonged shedding of BPZE1. Further evaluation of BPZE1 is warranted.     

Generation of growth arrested Leishmania amastigotes: A tool to develop live attenuated vaccine candidates against visceral leishmaniasis

Visceral leishmaniasis (VL) is fatal if not treated and is prevalent widely in the tropical and sub-tropical regions of world. VL is caused by the protozoan parasite Leishmania donovani or Leishmania infantum. Although several second generation vaccines have been licensed to protect dogs against VL, there are no effective vaccines against human VL [1]. Since people cured of leishmaniasis develop lifelong protection, development of live attenuated Leishmania parasites as vaccines, which can have controlled infection, may be a close surrogate to leishmanization. This can be achieved by deletion of genes involved in the regulation of growth and/or virulence of the parasite. Such mutant parasites generally do not revert to virulence in animal models even under conditions of induced immune suppression due to complete deletion of the essential gene(s). In the Leishmania life cycle, the intracellular amastigote form is the virulent form and causes disease in the mammalian hosts. We developed centrin gene deleted L. donovani parasites that displayed attenuated growth only in the amastigote stage and were found safe and efficacious against virulent challenge in the experimental animal models. Thus, targeting genes differentially expressed in the amastigote stage would potentially attenuate only the amastigote stage and hence controlled infectivity may be effective in developing immunity. This review lays out the strategies for attenuation of the growth of the amastigote form of Leishmania for use as live vaccine against leishmaniasis, with a focus on visceral leishmaniasis.     

Antibiotic resistance free plasmid DNA expressing LACK protein leads towards a protective Th1 response against Leishmania infantum infection

Canine visceral leishmaniasis is a serious public health concern in the Mediterranean basin since dogs are the main Leishmania infantum reservoir. However, there is not a vaccination method in veterinary use in this area, and therefore the development of a vaccine against this parasite is essential for the possible control of the disease. Previous reports have shown the efficacy of heterologous prime-boost vaccination with the pCIneo plasmid and the poxvirus VV (both Western Reserve and MVA strains) expressing L. infantum LACK antigen against canine leishmaniasis. As pCIneo-LACK plasmid contains antibiotic resistance genes, its use as a profilactic method is not recommended. Hence, the antibiotic resistance gene free pORT-LACK plasmid is a more suitable tool for its use as a vaccine. Here we report the protective and immunostimulatory effect of the prime-boost pORT-LACK/MVA-LACK vaccination tested in a canine experimental model. Vaccination induced a reduction in clinical signs and in parasite burden in the liver, an induction of the Leishmania-specific T cell activation, as well as an increase of the expression of Th1 type cytokines in PBMC and target organs.     

Extended safety studies of the attenuated live tuberculosis vaccine SO2 based on phoP mutant

Safety is one of the main concerns for attenuated live vaccine candidates. Here we extend the stability and attenuation studies of the promising tuberculosis vaccine candidate based on Mycobacterium tuberculosis phoP mutant strain, SO2. Stability of the phoP mutation was tested after sub-culturing SO2 strain for 6 months in laboratory media and also after 3 months of infection in SCID mice. Results showed no reversion of the phoP mutation either in vitro or in vivo. In addition, SO2 was fully sensitive to four major first-line antituberculous drugs against tuberculosis. Safety and toxicity studies were performed in guinea pigs. Animals were infected with a quantity of SO2 equivalent to 50 vaccination doses (2.5 × 10⁶ CFUs) and weight was monitored for 6 months. All animals survived and no histological lesions were found, showing full attenuation of SO2. Studies in a post-exposure model of guinea pigs and mice, previously infected with M. tuberculosis, were performed and no toxicity effects were found after inoculation of SO2. All these results together confirm that SO2 has a secure safety profile that encourages its use in clinical trials.     

Heterologous prime-boost immunization with live attenuated B. pertussis BPZE1 followed by acellular pertussis vaccine in mice

Pertussis is a severe and life-threatening infectious disease. Two successive generations of vaccines have strongly reduced its incidence over the last 70 years. However, despite excellent global vaccine coverage, it is still not under control and constitutes today the most frequent vaccine-preventable childhood disease. New vaccination approaches are therefore needed. Here, we provide preclinical proof of concept for a heterologous prime-boost strategy, using the live attenuated Bordetella pertussis vaccine candidate BPZE1 to prime infant and neonatal mice intranasally and a currently available acellular pertussis vaccine (aPV) as a booster. Intranasal vaccination with BPZE1 provided strong protection against challenge in neonatal mice, which could be boosted with a single dose of aPV. Furthermore, BPZE1 priming induced a strong Th1/Th17 response, which was maintained after repeated aPV administrations, in contrast to non-primed mice, in which aPV administrations resulted in Th2 skewing. In addition to T cell responses, intranasal administration of BPZE1 to infant or neonatal mice also primed antibody responses to B. pertussis antigens, with a strong preference of the IgG2a over the IgG1 isotypes, which was not seen in non-primed animals. Finally, neonatal BPZE1 priming strongly enhanced aPV-induced protection against B. pertussis challenge. These results lend support for a heterologous prime-boost strategy to control pertussis by using BPZE1 early in life and considering the current aPV administrations as booster vaccinations, thereby bridging the gap from birth to the first aPV immunizations and avoiding aPV-mediated Th2 skewing. A first-in-man clinical trial on BPZE1 has recently been successfully completed, which provides hope that these findings may be translated into human applications in the future.     

Influenza vaccination rates in children decline when the live attenuated influenza vaccine is not recommended

BackgroundIn 2016 the Centers for Disease Control and Prevention (CDC) recommended against using the live attenuated influenza vaccine (LAIV) for the 2016–2017 influenza season. This recommendation is potentially important for vaccination rates because perceived effectiveness and ease of administration are among the primary determinants of families decisions to vaccinate their children. This investigation sought to determine whether rates of pediatric influenza vaccination changed in a season when the LAIV was not recommended.MethodsThis study used cohort and cross sectional data from an academic primary care pediatric center in central Pennsylvania that serves approximately 12,500 patients. Early season (prior to November 1) and end-of-season (prior to March 1) vaccination rates in the 2015–16 and 2016–17 influenza seasons were recorded for individuals 2–17 years old. Repeat vaccination rates (percentage of children receiving influenza vaccination in one season who were also vaccinated in the next season) were recorded for the 2015–16 into 2016–17 seasons. A logistic regression model adjusting for race, ethnicity, age, insurance type and type of vaccination received was employed to identify predictors of repeat vaccination.ResultsIn the absence of LAIV (2016–17) early vaccination rates were significantly higher (24.7% vs 22.8%, p = 0.004), but end-of-season rates were lower (50.4% vs 52.0%, p = 0.03) than when LAIV was offered (2015–16). After adjusting for covariates, those who had received IIV in the 2015–16 season had higher odds (OR 1.32, 95% CI, 1.15–1.52) of getting a repeat vaccination in the 2016–17 season, compared with those who had received LAIV in the 2015–16 season.ConclusionsEnd-of-season vaccination rates were lower in 2016–17 when LAIV was not recommended, particularly among children who received LAIV in the preceding year. Unavailability of LAIV in the 2016–17 season may have impacted influenza vaccination convenience and perceived effectiveness, two factors which could influence vaccine uptake in pediatric populations.     

Development of live attenuated Streptococcus agalactiae as potential vaccines by selecting for resistance to sparfloxacin

To develop attenuated bacteria as potential live vaccines, sparfloxacin was used in this study to modify 40 isolates of Streptococcus agalactiae. Majority of S. agalactiae used in this study were able to develop at least 80-fold resistance to sparfloxacin. When the virulence of the sparfloxacin-resistant S. agalactiae isolates were tested in 10–12 g Nile tilapia by intraperitoneal injection at dose of 2 × 10⁷ CFU/fish, 31 were found to be avirulent to fish. Of the 31 avirulent sparfloxacin-resistant S. agalactiae isolates, 30 provided 75–100% protection to 10–12 g Nile tilapia against challenges with a virulent S. agalactiae isolate Sag 50. When the virulence of the 30 sparfloxacin-resistant S. agalactiae isolates was tested in 3–5 g Nile tilapia by intraperitoneal injection at dose of 2 × 10⁷ CFU/fish, six were found to be avirulent to 3–5 g Nile tilapia. Of the six avirulent sparfloxacin-resistant S. agalactiae isolates, four provided 3–5 g Nile tilapia 100% protection against challenges with homologous isolates, including Sag 97-spar isolate that was non-hemolytic. However, Sag 97-spar failed to provide broad cross-protection against challenges with heterologous isolates. When Nile tilapia was vaccinated with a polyvalent vaccine consisting of 30 sparfloxacin-resistant S. agalactiae isolates at dose of 2 × 10⁶ CFU/fish, the polyvalent vaccine provided significant (P < 0.001) protection to both 3–5 g and 15–20 g Nile tilapia against challenges with 30 parent isolates of S. agalactiae. Taken together, our results suggest that a polyvalent vaccine consisting of various strains of S. agalactiae might be essential to provide broader protection to Nile tilapia against infections caused by S. agalactiae.     

Mucosal vaccination with a codon-optimized hemagglutinin gene expressed by attenuated Salmonella elicits a protective immune response in chickens against highly pathogenic avian influenza

The purpose of this study was to evaluate clinical protection from challenge conferred by two attenuated Salmonella enteria serovar typhimurium vaccine strains expressing the hemagglutinin (HA1) gene from a highly pathogenic avian influenza (HPAI) H5N1 (A/whooper swan/Mongolia/3/2005), under control of the anaerobically inducible nir15 promoter. Two-week-old White Leghorn chickens were immunized by oral gavage with one milliliter doses of >109 Salmonella colony-forming units once weekly for 4 weeks prior to challenge. Expression of recombinant protein was confirmed via Western blot. Serum and mucosal gavage samples were collected prior to, and following immunization and antibodies against avian influenza HA were confirmed by Western blot and hemagglutination-inhibition (HI) assay. Chickens were challenged with homologous (A/whooper swan/Mongolia/3/2005), or heterologous (A/Chicken/Queretaro/14588-19/95) HPAI virus strains. Chickens immunized with attenuated Salmonella strains containing plasmid expression vector (pTETnir15HA) demonstrated a statistically significant increase in survival compared to control groups. Results provide evidence of effectiveness of attenuated Salmonella strains for delivery of recombinant avian influenza HA antigens and induction of mucosal and systemic immune responses protective against lethal challenge with HPAI.     

Towards an attenuated enterohemorrhagic Escherichia coli O157:H7 vaccine characterized by a deleted ler gene and containing apathogenic Shiga toxins

The aim of this study was to develop a candidate vaccine against enterohemorrhagic Escherichia coli O157:H7. A ler deletion mutant derived from wild-type EHEC O157:H7 86-24 was constructed by use of suicide vector pCVD442. The bacteriophage encoding Shiga toxin (Stx) was excised by serial passage to produce a ler/stx deletion mutant, F25. Stx1 and Stx2 mutants were constructed by site-directed mutagenesis within the active center and membrane-spanning region of the toxin A subunit. Mutants stx1 and stx2 were then introduced into F25 to construct live attenuated candidate vaccine F105. The cytotoxicity of F25 was inactivated and that of F105 was significantly reduced in comparison with wild-type E. coli strain EDL933. Mice injected with candidate vaccine strains F25 and F105 gained weight and showed no clinical signs of disease. F25 and F105 reduced the colonization of wild-type O157:H7 in mouse intestine. Immunized pregnant mice were able to protect their suckling newborns from intragastric challenge with wild-type O157:H7. Immunized mice were protected against infection with wild-type O157:H7 and exhibited normal weight gain. Such attenuated vaccine strains may therefore have potential use as oral vaccines against O157:H7.     

Evaluation of an attenuated strain of Ehrlichia canis as a vaccine for canine monocytic ehrlichiosis

Canine monocytic ehrlichiosis is an important tick-borne disease worldwide. No commercial vaccine for the disease is currently available and tick control is the main preventive measure against the disease. The aim of this study was to evaluate the potential of a multi-passaged attenuated strain of Ehrlichia canis to serve as a vaccine for canine monocytic ehrlichiosis, and to assess the use of azithromycin in the treatment of acute ehrlichiosis. Twelve beagle dogs were divided into 3 groups of 4 dogs. Groups 1 and 2 were inoculated (vaccinated) with an attenuated strain of E. canis (#611A) twice or once, respectively. The third group consisted of naïve dogs which served as controls. All 3 groups were challenged with a wild virulent strain of E. canis by administering infected dog-blood intravenously. Transient thrombocytopenia was the only hematological abnormality observed following inoculation of dogs with the attenuated strain. Challenge with the virulent strain resulted in severe disease in all 4 control dogs while only 3 of 8 vaccinated dogs presented mild transient fever. Furthermore, the mean blood rickettsial load was significantly higher in the control group (27–92-folds higher during days 14–19 post challenge with the wild the strain) as compared to the vaccinated dogs. The use of azithromycin was assessed as a therapeutic agent for the acute disease. Four days treatment resulted in further deterioration of the clinical condition of the dogs. Molecular comparison of 4 genes known to express immunoreactive proteins and virulence factors (p30, gp19, VirB4 and VirB9) between the attenuated strain and the challenge wild strain revealed no genetic differences between the strains. The results of this study indicate that the attenuated E. canis strain may serve as an effective and secure future vaccine for canine ehrlichiosis.     

KMP-11 DNA immunization significantly protects against L. donovani infection but requires exogenous IL-12 as an adjuvant for comparable protection against L. major

As vaccine potential of cross-species protection by a candidate antigen is less explored, in this study we compared cross-specific protective efficacy of Kinetoplastid Membrane Protein-11 (KMP-11) as a DNA vaccine alone and in conjunction with exogenous IL-12 administration in experimental BALB/c model against two most widely prevalent forms of clinical diseases caused by Leishmania major (LM) and Leishmania donovani (LD). Whereas, KMP-11 DNA vaccination alone showed significant potential in terms of resolution of splenic and hepatic parasite burden against virulent LD challenge, it showed considerably less efficacy (<70% reduction) against virulent LM challenge in terms of presence of parasite in lymph node. Remarkably exogenous IL-12 administration in the form of IL-12 p35/p40 expression vectors or recombinant protein along with KMP-11 DNA had exactly opposing effect on protection against LM and LD. Exogenous IL-12 administration significantly increased residual LD-burden but enhanced the protective efficacy of KMP-11 DNA vaccine against LM compared to KMP-11 immunization alone. Elucidation of effector mechanism showed KMP-11 DNA induced protection against LD was associated with the generation of mixed Th1/Th2 response, while KMP-11/IL-12-induced comparable protection against LM was associated with high IgG2a titre indicative of a polarized Th1 response. Exogenous IL-12 administration resulted in robust gamma interferon (IFN-γ) production and suppression of IL-4 from CD4⁺ T cell against both LM and LD. Nevertheless protective immune response was only compromised against LD infection where frequency of anti-KMP-11 CTL response was significantly reduced after exogenous IL-12 administration. Our study provides a comparative evaluation of effector mechanisms in the assessment of cross-specific protection by KMP-11 and KMP-11/IL-12 immunization against these two prevalent forms of leishmaniasis.     

Reactogenicity and immunogenicity of live attenuated Salmonella enterica serovar Paratyphi A enteric fever vaccine candidates

Eight Salmonella enterica serovar Paratyphi A strains were screened as candidates to create a live attenuated paratyphoid vaccine. Based on biochemical and phenotypic criteria, four strains, RKS2900, MGN9772, MGN9773 and MGN9779, were selected as progenitors for the construction of ΔphoPQ mutant derivatives. All strains were evaluated in vitro for auxotrophic phenotypes and sensitivity to deoxycholate and polymyxin B. All ΔphoPQ mutants were more sensitive to deoxycholate and polymyxin B than their wild-type progenitors, however MGN10028, MGN10044 and MGN10048, required exogenous purine for optimal growth. Purine requiring strains had acquired point mutations in purB during strain construction. All four mutants were evaluated for reactogenicity and immunogenicity in an oral rabbit model. Three strains were reactogenic in a dose-dependent manner, while one strain, MGN10028, was well-tolerated at all doses administered. All ΔphoPQ strains were immunogenic following a single oral dose. The in vitro profile coupled with the favorable reactogenicity and immunogenicity profiles render MGN10028 a suitable live attenuated Paratyphi A vaccine candidate.     

Immunotherapy with the saponin enriched-Leishmune vaccine versus immunochemotherapy in dogs with natural canine visceral leishmaniasis

Leishmune^®, the first licensed vaccine for prophylaxis against canine visceral leishmaniasis (CVL) and is also immunotherapeutic when used with double saponin adjuvant concentration. The Leishmune^® therapeutic vaccine was assessed for immunotherapy (IT) in 31 infected dogs and for immunochemotherapy (ICT) in combination with allopurinol or amphotericinB/allopurinol, in 35 dogs. Compared to infected untreated control dogs, at month 3, both treatments increased the proportion of dogs showing intradermal response to Leishmania antigen to a similar extent (from 8 to 67%, in the IT and to 76%, in the ICT groups), and conversely reduced from 100 to 38% (IT) and to 18% (ICT) the proportion of symptomatic cases, from 54 to 12% (IT) and to 15% (ICT) the proportion of parasite evidence in lymph nodes and from 48 to 19% (IT) and 12% (ICT) the proportion of deaths, indicating that the immunotherapy with enriched-Leishmune^® vaccine promotes the control of the clinical and parasitological signs of CVL rendering most dogs asymptomatic although PCR positive. By month 8, negative lymph node PCR results were obtained in 80% of the ICT-treated dogs, but only in 33% of the IT group (p = 0.0253), suggesting that the combination of additional chemotherapy with Leishmune^®-enriched saponin vaccination abolished, not only the symptoms but also the latent infection condition, curing the dogs. The animals were followed up until 4.5 years after the beginning of the experiment and, compared to the untreated control group at month 3 (12/25 dogs; 48%), a decrease in the rate of CVL deaths was only seen after ICT treatment (7/35 dogs; 20%; 0.0273) but not after IT treatment (10/31 dogs; 32%; p = 0.278), pointing out an additional advantage of the ICT treatment with the enriched-Leishmune^® in the control and cure of CVL.     

A vaccine candidate of attenuated genotype VII Newcastle disease virus generated by reverse genetics

Genotype VII Newcastle disease virus (NDV) has been documented as the predominant epidemic genotype in China and some other Asian countries since 1990s. Recent work has demonstrated that NDV vaccines phylogenetically closer to epidemic viruses provide better protection than conventional vaccines in terms of reducing virus shedding and transmission. Since there is currently no available vaccine which possesses a close antigenic relationship to the prevalent virulent NDV, a new vaccine to protect against the infection of this genotype NDV is in urgent need. Here, we describe the generation of a pathogenicity-attenuated genotype VII NDV (NDV/ZJ1HN) from a velogenic NDV by mutating the velogenic amino acid motif at the F protein cleavage site using reverse genetics techniques. The attenuated-pathogenicity of NDV/ZJ1HN was confirmed by examination of mean death time (MDT) in embryonated eggs and intracerebral pathogenicity index (ICPI) in day-old chickens. Subsequently, 2 weeks old birds were immunized with live and inactivated NDV/ZJ1HN-based vaccines and challenged 3 or 4 weeks post-immunization with a lethal dose of a virulent genotype VII NDV strain. Results showed that NDV/ZJ1HN effectively protected the vaccinated birds from morbidity and mortality against genotype VII virus challenge and significantly reduced virus shedding from the vaccinated birds when compared with La Sota vaccinated animals, suggesting that NDV/ZJ1HN is a promising vaccine candidate for the control of current ND epidemic in China.