Protection of cattle against a natural infection of Fasciola hepatica by vaccination with recombinant cathepsin L1 (rFhCL1)

The liver fluke, Fasciola hepatica causes liver fluke disease, or fasciolosis, in ruminants such as cattle and sheep. An effective vaccine against the helminth parasite is essential to reduce our reliance on anthelmintics, particularly in light of frequent reports of resistance to some frontline drugs. In our study, Friesian cattle (13 per group) were vaccinated with recombinant F. hepatica cathepsin L1 protease (rFhCL1) formulated in mineral-oil based adjuvants, Montanide™ ISA 70VG and ISA 206VG. Following vaccination the animals were exposed to fluke-contaminated pastures for 13 weeks. At slaughter, there was a significant reduction in fluke burden of 48.2% in the cattle in both vaccinated groups, relative to the control non-vaccinated group, at p ≤ 0.05. All vaccinated animals showed a sharp rise in total IgG levels to rFhCL1 post-vaccination which was maintained over the course of the 13-week challenge infection and was significantly higher than levels reached in the control group. Arginase levels in the macrophages of vaccinated cattle were significantly lower than those of the control cattle, indicating that the parasite-induced alternative-activation of the macrophages was altered by vaccination. The data demonstrate the potential for recombinant FhCL1 vaccine in controlling fasciolosis in cattle under field conditions.     

Contrasting patterns of population genetic structure of Fasciola hepatica from cattle and sheep: Implications for the evolution of anthelmintic resistance

Twelve polymorphic genetic markers, eight allozymic loci and four microsatellites, were used to characterize 20 infrapopulations of Fasciola hepatica (all flukes from 10 individual cattle and 10 sheep) from 11 farms in Northwest Spain. Results suggest different patterns of population genetic structure depending on the host species. Individuals identified as clones were much more frequent in sheep. The common presence of clones and its nonrandom occurrence among individual hosts suggests clumped transmission of liver flukes in sheep. After reducing significant repeated multilocus genotypes to one unique copy within infrapopulations, results show relatively high levels of gene diversity within infrapopulations from cattle and sheep (0.411 and 0.360 on average, respectively). However, parasites of sheep appear to show significantly more structured variation at the infrapopulation level (Standardized F_ST = 0.087 and 0.170 for parasites of cattle and sheep, respectively). Compared to the parasites from cattle, results suggest that populations from sheep show lower levels of gene flow, higher degree of aggregate transmission, higher probability of mating within clones, and lower parasitic load. These differences have implications for the evolution of anthelmintic resistance because they affect the effective population size and the degree of inbreeding. The development and rapid spread of resistance seems likely in the parasites of cattle because populations from the study area are characterized by high gene flow. However, results also suggest that the efficient selection of a new recessive advantageous mutation would be favored in parasites of sheep due to a greater potential for inbreeding.     

The recombinant gut-associated M17 leucine aminopeptidase in combination with different adjuvants confers a high level of protection against Fasciola hepatica infection in sheep

Fasciola hepatica M17 leucine aminopeptidase (FhLAP) is thought to play a role in catabolizing peptides generated by the concerted activity of gut-associated endopeptidases on host polypeptides, thus releasing amino acids to be used in protein anabolism. In this study, a recombinant functional form of this homo hexameric metallopeptidase produced in Escherichia coli was used in combination with adjuvants of different types in a vaccination trial in Corriedale sheep against experimental challenge with F. hepatica metacercariae. The experimental assay consisted of 6 groups of 10 animals; 5 of the groups (1-5) were subcutaneously inoculated at weeks 0 and 4 with100 μg of rFhLAP mixed with Freund's complete plus incomplete adjuvant (group 1), Alum (group 2), Adyuvac 50 (group 3), DEAE-D (group 4) and Ribi (group 5); the adjuvant control group (group 6) received Freund's adjuvant. Two weeks after the booster, the sheep were orally challenged with 200 metacercariae. Immunization with rFhLAP induced significant reduction in fluke burdens in all vaccinated groups: 83.8% in the Freund's group, 86.7% in the Alum group, 74.4% in the Adyuvac 50 group, 49.8% in the Ribi group and 49.5% in the DEAE-D group compared to the adjuvant control group. Morphometric analysis of recovered liver flukes showed no significant size modifications in the different vaccination groups. All vaccine preparations elicited specific IgG, IgG1 and IgG2 responses. This study shows that a liver fluke vaccine based on rFhLAP combined with different adjuvants significantly reduced worm burden in a ruminant species that was high in animals that received the enzyme along with the commercially approved adjuvants Alum and Adyuvac 50.     

Evaluation of humoral and cellular immune response of BALB/c mice immunized with a recombinant fragment of MSP1a from Anaplasma marginale using carbon nanotubes as a carrier molecule

Bovine anaplasmosis is a disease caused by the intraerythrocytic rickettsia Anaplasma marginale. Surface proteins (MSPs) of A. marginale are important in the interaction of the pathogen with the host and constitute potential vaccine targets against this pathogen. Currently, there is no commercial inactivated vaccine against bovine anaplasmosis that can generate a protective immune response that effectively prevents the development of clinical disease. The objective of this study was to evaluate the humoral and cellular immune responses of BALB/c mice immunized with the recombinant fragment of rMSP1a from A. marginale using carbon nanotubes as a carrier molecule. The fragment of rMSP1a comprising the N-terminal region of the protein was expressed in Escherichia coli BL21, purified by nickel affinity chromatography and covalently linked to multiwalled carbon nanotubes (MWNTs). After this functionalization, thirty BALB/c mice were divided into five groups, G1 (rMSP1a), G2 (MWNT + rMSP1a), G3 (MWNT), G4 (adjuvant) and G5 (unimmunized). The mice were immunized subcutaneously at days 0, 21 and 42. Blood samples were collected on day 11 after immunization. The spleens were collected, and the splenocytes were cultured for cell proliferation assays and cell immunophenotyping. Mice immunized with rMSP1a (G1 and G2) produced high levels of anti-rMSP1a IgG, demonstrating that the functionalization to carbon nanotubes did not interfere with protein immunogenicity. Immunization with MWNT + rMSP1a significantly induced higher percentages of CD4⁺CD44⁺ and CD4⁺CD62L⁺ lymphocytes, high levels of TNF-α, and a higher proliferative rate of splenocytes compared to mice immunized with rMSP1a alone (G1 group). Therefore, additional experiments using cattle should be performed to determine the efficacy, safety, immunogenicity and protection induced by rMSP1a associated with MWNT.     

Preputial cellular and antibody responses of bulls vaccinated and/or challenged with Tritrichomonas foetus

Systemic and genital immune responses in bulls were determined after infection with Tritrichomonas foetus and systemic vaccination with whole cell antigens. Vaccinated bulls resisted infection, developed IgG1 and IgG2 antibodies against T. foetus in preputial secretions and serum, and had increased MHC II⁺ and CD205⁺ cells (probably dendritic cells), CD3⁺ and CD8⁺ T cells, and B cells including IgG1 and IgA plasma cells in the prepuce. Non-vaccinated bulls challenged with T. foetus were persistently infected and had no detectable antibodies to T. foetus in either preputial secretions or serum for 6 weeks post challenge. We conclude that genital and serum IgG antibodies to T. foetus accounts for resistance of vaccinated bulls to T. foetus infection and that the lack of an antibody response in infected bulls accounts for persistent infection.     

Local and systemic immune responses in pigs intramuscularly injected with an inactivated Mycoplasma hyopneumoniae vaccine

The immune response induced by intramuscular administration of a commercial inactivated Mycoplasma hyopneumonie whole-cell vaccine (Suvaxyn^®MH One) was investigated in conventional M. hyopneumoniae-free pigs. The animals were assigned randomly to two groups: non-vaccinated and vaccinated. Pigs in the vaccinated group were injected intramuscularly with the vaccine at 7 days of age, whereas non-vaccinated pigs received physiological saline solution (PBS). Pigs were euthanized and necropsied at 30, 36 and 58 days of age. Blood, bronchoalveolar lavage (BAL) fluid, spleen, lung and bronchial lymph nodes (BLN) were collected. Serum and BAL fluid were tested for the presence of antibodies by ELISA. Monomorphonuclear cells from the peripheral blood and tissues were isolated to quantify the T cell subsets by flow cytometry, and cytokine production by ELIspot and ELISA. Antibodies against M. hyopneumoniae were detected in serum of most vaccinated pigs at 30 days of age. M. hyopneumoniae specific IgG, IgM and IgA were detected in BAL fluid from vaccinated animals, but not from control animals. Significantly higher numbers of IL-12 secreting cells were observed in the lung at day 58 in the vaccinated than in the non-vaccinated group (p < 0.05). The number of IL-10 secreting cells from BLN was also higher in the vaccinated group at day 58 (p < 0.05). After restimulation in vitro, lymphocytes from BLN and lungs secreted significantly higher levels of IL-12 in the vaccinated group at day 58. These results show that the vaccine induced both systemic and mucosal cellular and humoral immune responses.     

Long-lasting humoral and cellular immune responses elicited by immunization with recombinant chimeras of the Plasmodium vivax circumsporozoite protein

The circumsporozoite protein (CSP), the most abundant surface antigen of sporozoites, has been extensively studied in different expression platforms as a vaccine candidate. Clinical trials have shown the necessity of broad and highly avid humoral immune responses together with high numbers of CSP-specific T_CD4+ and T_CD8+ cells, especially those producing IFN-γ, to induce protection. To this aim, we designed two distinct recombinant immunogens based on previously-described antigenic fragments of Plasmodium vivax CSP (PvCSP) to be used as vaccine candidates. The first one is a virus-like particle (VLP) comprising the repeat region of PvCSP (B and T_CD4+ epitopes) within the loop of the hepatitis B virus core antigen (HBcAgPvCSP). The second one is a PvCSP multi-epitope polypeptide, rPvCSP-ME, designed based on antigenic regions of PvCSP recognized by lymphocytes of individuals from endemic areas.     

Construction and evaluation of replication-defective recombinant optimized triosephosphate isomerase adenoviral vaccination in Schistosoma japonicum challenged mice

Schistosomiasis is an endemic, zoonotic parasitic disease that remains a public health concern in China. Development of transmission blocking veterinary vaccines against Schistosoma japonicum infection is urgently needed. Replication-defective adenoviral vector is an efficient vaccine delivery system that has been widely used. Its use is associated with high levels of gene insertion and expression. It is easy to construct and prepare, and is safe. It is not known whether this delivery system can improve the protective effect of schistosome vaccination. Triosephosphate isomerase from S. japonicum (SjTPI) is a promising vaccine candidate. Thus far it has induced only partial protection in animal models and needs to be further enhanced to be effective. We constructed a replication-defective adenoviral vector-based vaccine with optimized SjTPI (rAdV-SjTPI.opt). The specific immune responses and protective efficiency in mice were evaluated. Results showed that intramuscular rAdV-SjTPI.opt induced Th1 biased immune responses in the host, while subcutaneous rAdV-SjTPI.opt induced Th2 predominant immune responses. Oral rAdV-SjTPI.opt induced low levels of immune responses and no significant protection. Intramuscular rAdV-SjTPI.opt provided a consistent and repeatable higher protective effect in mice (more than 50%). These findings may be due to the associated higher levels of specific Th1, antibody responses and partially lower level of IL-17A. This report provides a foundation for developing transmission-blocking veterinary vaccines in larger animals.     

Age-dependent systemic antibody responses and immunisation-associated changes in mice orally and nasally immunised with Lactococcus lactis expressing a malaria parasite protein

Gram positive food-grade bacteria such as lactococci have significant advantages over attenuated pathogens as vaccine delivery vehicles because of their inherently greater safety. Plasmodium falciparum merozoite surface antigen 2 (MSA2) was expressed in recombinant Lactococcus lactis both intracellularly and covalently anchored to the peptidoglycan of the cell wall (MSA2cP). Balb/c mice of different ages were immunised with the MSA2cP expressing L. lactis in a combined oral and nasal immunisation procedure. Serum IgG antibody responses to MSA2 were higher in young adult Balb/c mice compared to old mice and neonates. The elicited serum IgG antibodies reacted with native MSA2 on the surface of P. falciparum merozoites in an immunofluorescence assay. The serum IgG antibody isotypes in young adult mice were mainly IgG1, IgG2a and IgG2b, while IgG3 tended to be higher in old mice. IgA antibodies to MSA2 were also produced in young mice. Enlarged mesenteric lymph nodes, and more prominent lymphoid tissue in the lamina propria of the ileum and lymphoid follicles in the spleen, were observed in mice fed L. lactis. These findings are relevant for developing L. lactis as a vector to deliver vaccines in human populations.     

Vaccination of koalas (Phascolarctos cinereus) with a recombinant chlamydial major outer membrane protein adjuvanted with poly I:C,a host defense peptide and polyphosphazine,elicits strong and long lasting cellular and humoral immune responses

Chlamydial infections are wide spread in koalas across their range and a solution to this debilitating disease has been sought for over a decade. Antibiotics are the currently accepted therapeutic measure, but are not an effective treatment due to the asymptomatic nature of some infections and a low efficacy rate. Thus, a vaccine would be an ideal way to address this infectious disease threat in the wild. Previous vaccine trials have used a three-dose regimen; however this is very difficult to apply in the field as it would require multiple capture events, which are stressful and invasive processes for the koala. In addition, it requires skilled koala handlers and a significant monetary investment. To overcome these challenges, in this study we utilized a polyphosphazine based poly I:C and a host defense peptide adjuvant combined with recombinant chlamydial major outer membrane protein (rMOMP) antigen to induce long lasting (54 weeks) cellular and humoral immunity in female koalas with a novel single immunizing dose. Immunized koalas produced a strong IgG response in plasma, as well as at mucosal sites. Moreover, they showed high levels of C. pecorum specific neutralizing antibodies in the plasma as well as vaginal and conjunctival secretions. Lastly, Chlamydia-specific lymphocyte proliferation responses were produced against both whole chlamydial elementary bodies and rMOMP protein, over the 12-month period. The results of this study suggest that a single dose rMOMP vaccine incorporating a poly I:C, host defense peptide and polyphosphazine adjuvant is able to stimulate both arms of the immune system in koalas, thereby providing an alternative to antibiotic treatment and/or a three-dose vaccine regime.     

Evaluation of live-attenuated Salmonella vaccines expressing Campylobacter antigens for control of C. jejuni in poultry

Campylobacter jejuni is a zoonotic bacterial pathogen of worldwide importance. It is estimated that 460,000 human infections occur in the United Kingdom per annum and these involve acute enteritis and may be complicated by severe systemic sequelae. Such infections are frequently associated with the consumption of contaminated poultry meat and strategies to control C. jejuni in poultry are expected to limit pathogen entry into the food chain and the incidence of human disease. Toward this aim, a total of 840 Light Sussex chickens were used to evaluate a Salmonella enterica serovar Typhimurium ΔaroA vaccine expressing the C. jejuni amino acid binding protein CjaA as a plasmid-borne fusion to the C-terminus of fragment C of tetanus toxin. Chickens were given the vaccine at 1-day-old and two weeks later by oral gavage, then challenged after a further two weeks with C. jejuni. Across six biological replicates, statistically significant reductions in caecal C. jejuni of c. 1.4 log₁₀ colony-forming units/g were observed at three and four weeks post-challenge relative to age-matched unvaccinated birds. Protection was associated with the induction of CjaA-specific serum IgY and biliary IgA. Protection was not observed using a vaccine strain containing the empty plasmid. Vaccination with recombinant CjaA subcutaneously at the same intervals significantly reduced the caecal load of C. jejuni at three and four weeks post-challenge. Taken together these data imply that responses directed against CjaA, rather than competitive or cross-protective effects mediated by the carrier, confer protection. The impact of varying parameters on the efficacy of the S. Typhimurium ΔaroA vaccine expressing TetC-CjaA was also tested. Delaying the age at primary vaccination had little impact on protection or humoral responses to CjaA. The use of the parent strain as carrier or changing the attenuating mutation of the carrier to ΔspaS or ΔssaU enhanced the protective effect, consistent with increased invasion and persistence of the vaccine strains relative to the ΔaroA mutant. Expression in the ΔaroA strain of a TetC fusion to Peb1A, but not TetC fusions to GlnH or ChuA, elicited protection against intestinal colonisation by C. jejuni that was comparable to that observed with the TetC-CjaA fusion. Our data are rendered highly relevant by use of the target host in large numbers and support the potential of CjaA- and Peb1A-based vaccines for control of C. jejuni in poultry.     

Intraperitoneal immunization of recombinant HSP70 (DnaK) of Salmonella Typhi induces a predominant Th2 response and protective immunity in mice against lethal Salmonella infection

Heat shock proteins serve as important antigens in defense against infectious diseases. Members of HSP70 family, particularly microbial HSP70s have acquired special significance in immunity. In the present study, we evaluated the immunogenicity and protective efficacy of HSP70 of Salmonella enterica serovar Typhi against lethal infection by Salmonella in mice with or without adjuvants. The HSP70 gene was cloned and expressed in Escherichia coli BL21 and purified by affinity chromatography. Immunization of mice with HSP70 either alone or in combination with complete Freund's adjuvant (CFA) resulted in a significant increase in antibody titers as compared to control. Antibody isotyping revealed that HSP70 immunization induces both IgG1 and IgG2a antibodies to a significant extent but a higher IgG1/IgG2a ratio indicates a predominant Th2 response. There was a significant increase in lymphocyte proliferation, and levels of both Th2 and Th1 cytokines in cells isolated from immunized mice as compared to control. Immunization of mice with recombinant HSP70 either alone or in combination with CFA conferred 70-90% protection against lethal infections by Salmonella Typhi Ty2 or Salmonella Typhimurium. However, passive immunization with anti-HSP70 sera induced only partial protection in the immunized mice.     

Characterization of possible correlates of protective response against Brucella ovis infection in rams immunized with the B. melitensis Rev 1 vaccine

Vaccination with the live attenuated Brucella melitensis Rev 1 vaccine is used to control ovine brucellosis caused by Brucella ovis in sheep. The objective of this study was to identify possible correlates of protective response to B. ovis infection through the characterization by microarray hybridization and real-time RT-PCR of inflammatory and immune response genes differentially expressed in rams previously immunized with B. melitensis Rev 1 and experimentally challenged with B. ovis. Gene expression profiles were compared before and after challenge with B. ovis between rams protected and those vaccinated but found infected after challenge. The TLR10, Bak and ANXI genes were expressed at higher levels in vaccinated and protected rams. These genes provide possible correlates of protective response to B. ovis infection in rams immunized with the B. melitensis Rev 1 vaccine.     

Innate and acquired immune responses induced by recombinant Lactobacillus casei displaying flagellin-fusion antigen on the cell-surface

Bacterial flagellins are known as antigens that induce innate immune responses through TLR5 and boost immune responses in combination with other antigens. The aim of the present study was to determine the immunological properties of recombinant Lactobacillus casei producing flagellin and flagellin-fusion antigens in vitro and in vivo. Recombinant lactobacilli expressing Salmonella FliC and FliC fused to truncated SipC on the cell-surface were constructed. Fusion and non-fusion flagellin associated with L. casei retained the ability to induce IL-8 production by Caco-2 cells. Immunization of mice with these recombinant strains induced antigen-specific antibodies and cytokine production. The results showed that the outside epitope of the heterologous antigen was recognized more easily by the immune system than the inside epitope. The immune responses elicited by the Lactobacillus-associated antigens were mainly Th1 while that by the soluble antigen was Th2, although some of the responses were mixed.     

Antigen-specific B memory cell responses to lipopolysaccharide (LPS) and invasion plasmid antigen (Ipa) B elicited in volunteers vaccinated with live-attenuated Shigella flexneri 2a vaccine candidates

We evaluated B memory responses in healthy adult volunteers who received one oral dose of live-attenuated Shigella flexneri 2a vaccine. LPS-specific B_M cells increased from a median of 0 at baseline to 20 spot forming cells (SFC)/10⁶ expanded cells following vaccination (p = 0.008). A strong correlation was found between post-vaccination anti-LPS B_M cell counts and peak serum anti-LPS IgG titers (rs = 0.95, p = 0.0003). Increases in B_M specific for IpaB approaching significance were also observed. In sum, oral vaccination with live-attenuated S. flexneri 2a elicits B_M cells to LPS and IpaB, suggesting that B_M responses to Shigella antigens should be further studied as a suitable surrogate of protection in shigellosis.     

Characterization of protective immune responses promoted by human antigen targets in a urogenital Chlamydia trachomatis mouse model

A vaccine against genital tract infections caused by Chlamydia trachomatis is urgently needed. We have previously identified a number of immunodominant human T- and/or B-cell antigen targets in patients with a C. trachomatis infection. Herein we use a urogenital C. trachomatis mouse model to investigate the protective efficacy of these antigens. C3H/HeN mice were immunized with recombinant antigens formulated in the adjuvant CAF01. Immunity post vaccination was analyzed and the protective efficacy against vaginal challenge with C. trachomatis was monitored by vaginal swabbing. All antigens elicited a significant immune response when administered in CAF01 but the balance between CMI and humoral responses differed markedly for the different antigens. Six (CT443, CT043, CT858, CT610, CT004 and CT681) antigens were found to be protective. We demonstrated by T-cell depletion studies that the protection promoted by the two antigens CT043 and CT004 was mediated by CD4⁺ T-cells. Both antigens are frequently recognized by T-cells during a natural Chlamydia infection in humans and if included in a future multi-component Chlamydia vaccine probably would operate mainly through the induction of a CMI response.     

Evaluation of humoral and cellular immune responses to BP26 and OMP31 epitopes in the attenuated Brucella melitensis vaccinated sheep

In recent years, the number of cases of human brucellosis has been increasing by approximately 10% per year in China. Most cases were caused by Brucella melitensis through contacts with infected sheep, goats or their products. An attenuated B. melitensis vaccine M5-90 is currently used to vaccinate both animals in China. This vaccine has not been investigated for critical parameters such as immune response and its association with protective efficacy. In this study, humoral and cellular immune response to the periplasmic protein BP26 and the outer membrane protein OMP31 were evaluated in M5-90 vaccinated Chinese merino and Kazak sheep. Antibodies to BP26 or OMP31 were detected at low levels, and specific IFN-γ response was quantified. Strongly reactive peptides derived from BP26 and OMP31 identified five T-cell epitopes (BP26-6, -8, -11, -12 and OMP31-23) common to both sheep species, five species-specific epitopes (BP26-10, -18, -21 and -22 and OMP31-12) and four animal-specific epitopes (BP26-15, -23, OMP31-6 and -21), which stimulated specific IFN-γ response in vaccinated sheep. Among those T-cell epitopes, reactivity to BP26-18 and -21 epitopes was significantly associated with MHC-I B allele (P = 0.024). However, a specific T-cell response induced by the M5-90 vaccine was relatively week and did not sustain long enough, which might be suppressed by rapid activation of T-regulatory (Treg) cells following vaccination. These findings provide an insight in designing a safer and more effective vaccine for use in animals and in humans.