Immunological Considerations for Schistosoma Vaccine Development: Transitioning to Endemic Settings.

Despite mass drug administration programmes with praziquantel, the prevalence of schistosomiasis remains high. A vaccine is urgently needed to control transmission of this debilitating disease. As some promising schistosomiasis vaccine candidates are moving through pre-clinical and clinical testing, we review the immunological challenges that these vaccine candidates may encounter in transitioning through the clinical trial phases in endemic settings. Prior exposure of the target population to schistosomes and other infections may impact vaccine response and efficacy and therefore requires considerable attention. Schistosomes are known for their potential to induce T-reg/IL-10 mediated immune suppression in populations which are chronically infected. Moreover, endemicity of schistosomiasis is focal whereby target and trial populations may exhibit several degrees of prior exposure as well as in utero exposure which may increase heterogeneity of vaccine responses. The age dependent distribution of exposure and development of acquired immunity, and general differences in the baseline immunological profile, adds to the complexity of selecting suitable trial populations. Similarly, prior or concurrent infections with other parasitic helminths, viral and bacterial infections, may alter immunological responses. Consequently, treatment of co-infections may benefit the immunogenicity of vaccines and may be considered despite logistical challenges. On the other hand, viral infections leave a life-long immunological imprint on the human host. Screening for serostatus may be needed to facilitate interpretation of vaccine responses. Co-delivery of schistosome vaccines with PZQ is attractive from a perspective of implementation but may complicate the immunogenicity of schistosomiasis vaccines. Several studies have reported PZQ treatment to induce both transient and long-term immuno-modulatory effects as a result of tegument destruction, worm killing and subsequent exposure of worm antigens to the host immune system. These in turn may augment or antagonize vaccine immunogenicity. Understanding the complex immunological interactions between vaccine, co-infections or prior exposure is essential in early stages of clinical development to facilitate phase 3 clinical trial design and implementation policies. Besides well-designed studies in different target populations using schistosome candidate vaccines or other vaccines as models, controlled human infections could also help identify markers of immune protection in populations with different disease and immunological backgrounds.

Resistance to Chronic Toxoplasma gondii Infection Induced by a DNA Vaccine Expressing GRA16.

Toxoplasma gondii can infect all warm-blooded animals including human beings. T. gondii dense granule protein 16 (TgGRA16) as a crucial virulence factor could modulate the host gene expression. Here, a DNA vaccine expressing TgGRA16 was constructed to explore the protective efficacy against T. gondii infection in Kunming mice. The immune responses induced by pVAX-GRA16 were also evaluated. Mice immunized with pVAX-GRA16 could elicit higher levels of specific IgG antibody and strong cellular response compared to those in controls. The DNA vaccination significantly increased the levels of cytokines (IFN-γ, IL-2, IL-4, and IL-10) and the percentages of CD4+ and CD8+ T cells in mice. After lethal challenge, mice immunized with pVAX-GRA16 (8.4 ± 0.78 days) did not show a significant longer survival time than that in controls (7.1 ± 0.30 days) (p > 0.05). However, in chronic toxoplasmosis model (administration of 10 brain cysts of PRU strain orally), numbers of tissue cysts in mice immunized with pVAX-GRA16 were significantly reduced compared to those in controls (p < 0.05) and the rate of reduction could reach 43.89%. The results indicated that the TgGRA16 would be a promising vaccine candidate for further development of effective epitope-based vaccines against chronic T. gondii infection in mice.

Methods to Evaluate the Preclinical Safety and Immunogenicity of Genetically Modified Live-Attenuated Leishmania Parasite Vaccines.

Live-attenuated parasite vaccines are being explored as potential vaccine candidates since other approaches of vaccination have not produced an effective vaccine so far. In order for live-attenuated parasite vaccines to be tested in preclinical studies and possibly in clinical studies, the safety and immunogenicity of these organisms must be rigorously evaluated. Here we describe methods to test persistence in the immunized host and immunogenicity, and to identify biomarkers of vaccine safety and efficacy with particular reference to genetically attenuated Leishmania parasites.

Immunological comparison of DNA vaccination using two delivery systems against canine leishmaniasis.

Visceral leishmaniasis (VL) is a fatal disease caused by the intracellular protozoan parasite Leishmania infantum. Dogs are the primary reservoirs of this parasite, and vaccination of dogs could be an effective method to reduce its transfer to humans. In order to develop a vaccine against VL (apart from the choice of immunogenic candidate antigens), it is necessary to use an appropriate delivery system to promote a proper antigen-specific immune response. In this study, we compared two vaccine delivery systems, namely electroporation and cationic solid-lipid nanoparticle (cSLN) formulation, to administer a DNA vaccine containing the Leishmania donovani A2 antigen, and L. infantum cysteine proteinases of type I (CPA) and II (CPB) without its unusual C-terminal extension. The protective potencies of these two vaccine delivery systems were evaluated against L. infantum challenge in outbred dogs. Our results show that the administration of pcDNA-A2-CPA-CPB(-CTE)GFP vaccine as a prime-boost by either electroporation or cSLN formulation protects the dogs against L. infantum infection. Partial protection in vaccinated dogs is associated with significantly (p<0.05) higher levels of IgG2, IFN-γ, and TNF-α and with low levels of IgG1 and IL-10 as compared to the control group. Protection was also correlated with a low parasite burden and a strong delayed-type hypersensitivity (DTH) response. This study demonstrates that both electroporation and cSLN formulation can be used as efficient vaccine delivery systems against visceral leishmaniasis.

Supplemental dietary L-arginine attenuates intestinal mucosal disruption during a coccidial vaccine challenge in broiler chickens.

The present study investigated the effects of dietary arginine (Arg) supplementation on intestinal structure and functionality in broiler chickens subjected to coccidial challenge. The present study was a randomised complete block design employing a 3 × 2 factorial arrangement (n 8) with three dietary concentrations of Arg (11·1, 13·3 and 20·2 g/kg) with or without coccidial vaccine challenge (unchallenged and coccidial challenge). On day 14, birds were orally administered with coccidial vaccine or saline. On day 21, birds were killed to obtain jejunal tissue and mucosal samples for histological, gene expression and mucosal immunity measurements. Within 7 d of the challenge, there was a decrease in body-weight gain and feed intake, and an increase in the feed:gain ratio (P< 0·05). Jejunal inflammation was evidenced by villus damage, crypt dilation and goblet cell depletion. Coccidial challenge increased mucosal secretory IgA concentration and inflammatory gene (iNOS, IL-1ß, IL-8 and MyD88) mRNA expression levels (P< 0·05), as well as reduced jejunal Mucin-2, IgA and IL-1RI mRNA expression levels (P< 0·05). Increasing Arg concentration (1) increased jejunal villus height (P< 0·05) and linearly increased jejunal crypt depth (P< 0·05); (2) quadratically increased mucosal maltase activity (P< 0·05) and linearly decreased mucosal secretory IgG concentration (P< 0·05) within the coccidiosis-challenged groups; and (3) linearly decreased jejunal Toll-like receptor 4 (TLR4) mRNA expression level (P< 0·05) within the coccidiosis-challenged groups. The mRNA expression of mechanistic target of rapamycin (mTOR) complex 1 pathway genes (mTOR and RPS6KB1) and the anti-apoptosis gene Bcl-2 quadratically responded to increasing dietary Arg supplementation (P< 0·05). These results indicate that dietary Arg supplementation attenuates intestinal mucosal disruption in coccidiosis-challenged chickens probably through suppressing TLR4 and activating mTOR complex 1 pathways.

Attenuation of Theileria lestoquardi infected cells and immunization of sheep against malignant ovine theileriosis.

Malignant ovine theileriosis caused by Theileria lestoquardi is an economically important disease infecting small ruminants in the Sudan. The disease causes massive losses among sheep in many regions of Northern Sudan. The present studies were done to isolate lymphoblastoid cells infected with malignant ovine theileriosis and attenuate them by passage using culture media to develop and produce schizonts candidate vaccine, then test its efficacy and safety by exposing immunized lambs to field challenge in an area endemic with T. lestoquardi. In the present experiments we isolated and established an in vitro culture of T. lestoquardi infected lymphoblast cell line. Long-term culture of T. lestoquardi infected lymphoplastoid cells was shown to result in attenuation of their virulence and lambs inoculated with different doses of such cells at passage 105 exhibited very mild reactions with fever that lasted for 1-5 days and parasitaemia of <0.2%. The experimental lambs immunized with this candidate vaccine were immune and protected when exposed to field challenge in an area endemic of ovine theileriosis, while morbidity and mortality among non-immunized animals reached 76.9% and 46.15%, respectively, and they exhibited the clinical signs of malignant ovine theileriosis that included, high fever, loss of appetite, enlargement of lymph nodes, jaundice, loss of weight and death. The present study demonstrates the efficacy and the safety of this attenuated cell line as a live attenuated candidate vaccine.

Maternal antibody parameters of cattle and calves receiving EG95 vaccine to protect against Echinococcus granulosus.

Cattle may act as hosts for the transmission of the cestode parasite Echinococcus granulosus and play a role in transmission of the parasite leading to human cystic echinococcosis (CE). The recombinant EG95 vaccine has been shown to be able to protect cattle and other intermediate host species against CE. Ideally the immunisation of bovines against E. granulosus, using EG95 vaccine, should occur early in life so as to provide maximum protection against the establishment of hydatid cysts. Maternally derived antibody from vaccinated cows may provide some protection for the neonate, but may also interfere with the active response to vaccination. Experiments were undertaken to determine the optimal regime for protection of young cattle against CE. One group of pregnant cattle received 2 vaccinations of EG95 antigen+Quil A adjuvant two months and one month prior to calving. The control group of pregnant cattle were not vaccinated. Calves were either challenged with E. granulosus eggs at 4, 9, 13 or 17 weeks post-birth or were given their first vaccination at 8, 12 or 16 weeks post-birth. Sera obtained at regular intervals were tested by ELISA to assess the immunological response. All calves were experimentally challenged with E. granulosus eggs and subsequent necropsy confirmed the levels of protection. Maternal antibody was shown to protect calves to some extent for at least 17 weeks. Calves from vaccinated cows responded well serologically if the first vaccination was given at 8 or 12 weeks, but full protection against a challenge infection was achieved only if the first vaccination was delayed until 16 weeks after birth. Calves from non-vaccinated cattle also were not fully protected if the first vaccination was at 8 or 12 weeks, but were fully protected if the first vaccination was given when they were 16 weeks old. This suggests that immunological maturity is not acquired in calves until 4 or 5 months of age. No safety problems were observed following two vaccinations of 40 pregnant cows or 30 suckling calves.

Chemokine receptor CCR7 and CXCR5 mRNA in chickens following inflammation or vaccination.

The CCR7 and CXCR5 chemokine receptor mRNA contents of different immune organs were studied in normal, healthy birds and in birds treated with either lipopolysaccharide (LPS) as a systemic inflammatory challenge or coccidial vaccine (Coccivac B; Intervet/Schering-Plough Animal Health Corp., Millsboro, DE) as an enteric vaccination challenge. The CCR7 mRNA content of the spleen of normal, healthy birds was approximately 150-fold higher than CCR7 mRNA content of any other organs studied. The CXCR5 mRNA content of the bursa of normal, healthy birds was approximately 80-fold higher than the CXCR5 mRNA content of any other organs studied. The LPS injection decreased the splenic CCR7 mRNA content by approximately 100 times and the bursal CXCR5 mRNA content by approximately 5-fold at 24 h post-LPS injection (P < 0.01). The LPS injection increased the CXCR5 content of cecal tonsils by approximately 3-fold at 24 h post-LPS injection (P < 0.05). At 10 d postvaccination, CCR7 mRNA content was approximately 15-fold higher and CXCR5 mRNA content was approximately 12-fold higher in cecal tonsils of the vaccinated group than in the control group (P < 0.01). In conclusion, CCR7 and CXCR5 mRNA levels were dependent on the immune organs and the inflammatory status of the organs in chickens.

A clinical trial to evaluate the safety and immunogenicity of the LEISH-F1+MPL-SE vaccine when used in combination with meglumine antimoniate for the treatment of cutaneous leishmaniasis.

Forty-four adult patients with cutaneous leishmaniasis (CL) were enrolled in a randomized, double-blind, controlled, dose-escalating clinical trial and were randomly assigned to receive three injections of either the LEISH-F1+MPL-SE vaccine (consisting of 5, 10, or 20 µg recombinant Leishmania polyprotein LEISH-F1 antigen+25 µg MPL-SE adjuvant) (n=27), adjuvant alone (n=8), or saline placebo (n=9). The study injections were given subcutaneously on Days 0, 28, and 56, and the patients were followed through Day 336 for safety, immunological, and clinical evolution endpoints. All patients received chemotherapy with meglumine antimoniate starting on Day 0. The vaccine was safe and well tolerated. Nearly all vaccine recipients and no adjuvant-alone or placebo recipients demonstrated an IgG antibody response to LEISH-F1 at Day 84. Also at Day 84, 80% of vaccine recipients were clinically cured, compared to 50% and 38% of adjuvant-alone and placebo recipients. The LEISH-F1+MPL-SE vaccine was safe and immunogenic in CL patients and appeared to shorten their time to cure when used in combination with meglumine antimoniate chemotherapy.

Mic1-3KO tachyzoite a live attenuated vaccine candidate against toxoplasmosis derived from a type I strain shows features of type II strain.

Vaccination with live attenuated parasites has been shown to induce high level of protection against Toxoplasma gondii. In this study we compared the Mic1-3KO tachyzoite (a live attenuated strain) with the parental wild type (WT) tachyzoite in terms of virulence in mice in vivo, dissemination in mouse tissues and persistence in mouse brain. Survival of mice infected with the Mic1-3KO parasites correlated with reduced parasite burden in mouse tissues compared to the parental strain. Like the WT parasite, Mic1-3KO is able to form tissue cysts in vivo which are not, in our experimental conditions, infectious when given by oral route. Infection with the attenuated tachyzoite induced lower levels of cytokine and chemokine than with the parental strain. These data demonstrate that the deleted strain derived from a type I strain behaves like type II strain in outbred mice in terms of virulence, dissemination in mouse tissue and persistence in brain.

Effective humoral and cellular immunoprotective responses in Li ESAp-MDP vaccinated protected dogs.

Cell-mediated and humoral immunity were explored in LiESAp-MDP vaccinated protected dogs versus susceptible placebo dogs 2 months and 8 months post-vaccination. As previously described, a strong and long-lasting cell-mediated immunity, critical for protection against Leishmania infantum was exclusively revealed in vaccinated dogs as confirmed by a positive response to the intradermal inoculation of leishmanin and by a significant higher anti-leishmanial activity of canine monocytes-derived macrophages. Moreover, our results support the view that cooperation of humoral antibody with cell-mediated immunity might be important in developing protective immunity in LiESAp-MDP vaccinated dogs. Anti-LiESAp serum samples were found functionally active in vitro, promoting (i) early killing of pretreated promastigotes and amastigotes, (ii) strong inhibitory effect on the in vitro growth of both parasitic developmental stages of L. infantum and (iii) most importantly, a significant inhibition of pretreated promastigotes in vitro infectivity in canine macrophages. However, anti-LiESAp antibody response was not implicated in the promastigotes-amastigotes differentiation process. In these experiments, we have added additional support to the concept that antibodies to Leishmania may be important in developing protective immunity.

MVA-LACK as a safe and efficient vector for vaccination against leishmaniasis.

An optimal vaccine against leishmaniasis should elicit parasite specific CD4+ and cytotoxic CD8+ T cells. In this investigation, we described a prime/boost immunization approach based on DNA and on poxvirus vectors (Western Reserve, WR, and the highly attenuated modified vaccinia virus Ankara, MVA), both expressing the LACK antigen of Leishmania infantum, that triggers different levels of specific CD8+ T cell responses and protection (reduction in lesion size and parasitemia) against L. major infection in mice. A prime/boost vaccination with DNA-LACK/MVA-LACK elicits higher CD8+ T cell responses than a similar protocol with the replication competent VV-LACK. Both CD4+ and CD8+ T cells were induced by DNA-LACK/MVA-LACK immunization. The levels of IFN-gamma and TNF-alpha secreting CD8+ T cells were higher in splenocytes from DNA-LACK/MVA-LACK than in DNA-LACK/VV-LACK immunized animals. Moreover, protection against L. major was significantly higher in DNA-LACK/MVA-LACK than in DNA-LACK/VV-LACK immunized animals when boosted with the same virus dose, and correlated with high levels of IFN-gamma and TNF-alpha secreting CD8+ T cells. In DNA-LACK/MVA-LACK vaccinated animals, the extent of lesion size reduction ranged from 65 to 92% and this protection was maintained for at least 17 weeks after challenge with the parasite. These findings demonstrate that in heterologous prime/boost immunization approaches, the protocol DNA-LACK/MVA-LACK is superior to DNA-LACK/VV-LACK in triggering specific CD8+ T cell immune responses and in conferring protection against cutaneous leishmaniasis. Thus, MVA-LACK is a safe and efficient vector for vaccination against leishmaniasis.

Treatment regimens for patients with toxoplasmic encephalitis.

Toxoplasma gondii is an obligate intracellular parasitic protozoan that infects a variety of warm-blooded animals, including humans. Infection is usually asymptomatic in immunocompetent individuals but may be devastating in immunocompromised individuals such as those with acquired immunodeficiency syndrome (AIDS). Clinical manifestations of infection in immunocompromised patients include the development of encephalitis. It has been estimated that approximately 30% of patients with AIDS who are latently infected will eventually develop toxoplasmic encephalitis. The most common regimen used to treat toxoplasmic encephalitis is a combination of pyrimethamine 50 to 100 mg/d and sulfadiazine 4 to 8 g/d, with or without folinic acid 10 mg/d. This regimen, however, commonly leads to adverse effects or relapses. Other pharmacologic approaches include the use of clindamycin rather than sulfadiazine, the macrolide antibiotics, atovaquone, 5-fluorouracil, trimethoprim/sulfamethoxazole, minocycline or doxycycline, trimetrexate with folinic acid, dapsone, rifabutin, pentamidine, and diclazuril. None of these alternative regimens has been proven to be more effective than the standard pharmacologic therapy. An evolving approach is the use of immunotherapy, such as interleukin-2, -6, and -12; interferon-gamma; and alpha-tumor necrosis factor. Restoring a competent immune system may be the only cure for toxoplasmosis and other opportunistic infections.

Intact immune defenses are required for mice to resist the ts-4 vaccine strain of Toxoplasma gondii.

The ts-4 strain of Toxoplasma gondii is a temperature-sensitive mutant that fails to grow at 40 degrees C in vitro. Unlike mildly virulent cyst-forming strains, which can cause fatal chronic infections in certain mouse strains, ts-4 has been widely used to vaccinate mice against virulent T. gondii and is a valuable tool with which to investigate mechanisms of acquired resistance to this parasite. In this report, the basis for the avirulence of ts-4 is analyzed. It is shown that ts-4 is able to persist long-term in vivo in mildly immunocompromised mice, which rules out an intrinsic growth defect as a reason for avirulence. ts-4 does not induce body temperatures in mice as high as that needed to kill it in vitro. Moreover, the mild fevers elicited in resistant B6 mice are also seen in susceptible C57BL/6 scid/scid mice. However, ts-4 elicits strong preimmune defenses, dependent on gamma interferon, which are needed by mice to survive acute infection. Furthermore, CD4+ and CD8+ T-cell-dependent acquired immunity is essential for long-term survival of ts-4-infected mice.

Use of adjuvant containing mycobacterial cell-wall skeleton, monophosphoryl lipid A, and squalane in malaria circumsporozoite protein vaccine.

Human immune responses to modern synthetic and recombinant peptide vaccines administered with the standard adjuvant, aluminum hydroxide, tend to be poor, hence the search for better adjuvants. Antibody responses to a Plasmodium falciparum circumsporozoite (CS) protein vaccine, R32NS1(81), administered with an adjuvant containing cell-wall skeleton of mycobacteria and monophosphoryl lipid A in squalane (MPL/CWS) have been compared to responses to the same immunogen administered with aluminum hydroxide. 2 weeks after the third dose the following indices were greater in the 5 patients who received MPL/CWS than in controls (p less than 0.05): the geometric mean concentration (2.0 vs 25.4 microgram/ml) and avidity index of antibodies to the P falciparum CS protein by ELISA, the geometric mean titre to P falciparum sporozoites by IFAT (1/115 vs 1/1600), and the geometric mean inhibition of sporozoite invasion of hepatoma cells in vitro (37.6 vs 90.3%). For R32NS1(81) MPL/CWS is superior to aluminum hydroxide as an adjuvant, and the data support the evaluation of this complex as an adjuvant for other vaccines.