Immune response and protective efficacy of two new adjuvants, Montanide™ ISA 763B VG and Montanide™ GEL02, administered with a Streptococcus agalactiae ghost vaccine in Nile tilapia (Oreochromis niloticus).

Streptococcus agalactiae is one of the most important pathogens infecting tilapia worldwide and causes meningoencephalitis, septicemia and high mortalities with considerable losses. Various types of vaccines have been developed against S. agalactiae infection, such as inactivated vaccines, live attenuated vaccines and subunit vaccines. Bacterial ghosts (BGs) are nonliving, empty cell envelopes and have been reported as novel vaccine candidates. Therefore, the main aims of this study were to develop an S. agalactiae ghost vaccine (SAGV) and to evaluate the immune response and protective effect of SAGV against S. agalactiae with two novel adjuvants, Montanide™ ISA 763B VG and Montanide™ GEL02. Nile tilapia, mean weight 50 g, were divided into four groups as follows; 1) fish injected with PBS as control, 2) fish injected with the SAGV alone; 3) fish injected with the SAGV+Montanide™ ISA 763B VG; and 4) fish injected with SAGV+Montanide™ GEL02. Following vaccination, innate immunity parameters including serum lysozyme, myeloperoxidase, catalase, and bactericidal activity were all significantly enhanced. Moreover, specific serum IgM antibodies were induced and reached their highest level 2-8 weeks post vaccination. Importantly, the relative percent survival of tilapia vaccinated against the SAGV formulated with both adjuvants was 80-93%. Furthermore, the transcription of immune-related genes (IgM, TCRß, IL-1ß, IL-8 and TNFα) were up-regulated in tilapia after vaccination, indicating that both cellular and humoral immune responses were induced by these adjuvanted vaccines. In summary, Montanide™ ISA 763B VG and Montanide™ GEL02 can enhance immunoprotection induced by the SAGV vaccine against streptococcosis, demonstrating that both have value as potential adjuvants of fish vaccines.

A New Adjuvant Combined with Inactivated Influenza Enhances Specific CD8 T Cell Response in Mice and Decreases Symptoms in Swine Upon Challenge.

Vaccination is the most effective way to control swine influenza virus (SIV) in the field. Classical vaccines are based on inactivated antigens formulated with an oil emulsion or a polymeric adjuvant. Standard adjuvants enhance the humoral response and orient the immune response toward a Th2 response. An important issue is that current vaccines do not protect against new strains. One approach to improve cross-protection is to enhance Th1 and cytotoxic responses. The development of adjuvants orienting the immune response of inactivated vaccines toward Th1/Cytotoxic responses would be highly beneficial. This study shows that the water in oil in water emulsion adjuvant Montanide™ ISA 201 VG allows the induction of anti-influenza CD8 T cell in mice and induces homologous protection against an H1N1 challenge in swine. Such adjuvants that induce both humoral and cell-mediated immunity could improve the protection conferred by SIV vaccines in the field.

Evaluation of Montanide™ ISA 71 VG adjuvant during profilin vaccination against experimental coccidiosis.

Chickens were immunized subcutaneously with an Eimeria recombinant profilin protein plus Montanide™ ISA 70 VG (ISA 70) or Montanide™ ISA 71 VG (ISA 71) water-in-oil adjuvants, or with profilin alone, and comparative RNA microarray hybridizations were performed to ascertain global transcriptome changes induced by profilin/ISA 70 vs. profilin alone and by profilin/ISA 71 vs. profilin alone. While immunization with profilin/ISA 70 vs. profilin alone altered the levels of more total transcripts compared with profilin/ISA 71 vs. profilin alone (509 vs. 296), the latter was associated with a greater number of unique biological functions, and a larger number of genes within these functions, compared with the former. Further, canonical pathway analysis identified 10 pathways that were associated with genes encoding the altered transcripts in animals immunized with profilin/ISA 71 vs. profilin alone, compared with only 2 pathways in profilin/ISA 70 vs. profilin alone. Therefore, ISA 71 was selected as a candidate adjuvant in conjunction with profilin vaccination for in vivo disease protection studies. Vaccination with profilin/ISA 71 was associated with greater body weight gain following E. acervulina infection, and decreased parasite fecal shedding after E. maxima infection, compared with profilin alone. Anti-profilin antibody levels were higher in sera of E. maxima- and E. tenella-infected chickens vaccinated with profilin/ISA 71 compared with profilin alone. Finally, the levels of transcripts encoding interferon-γ, interleukin (IL)-2, IL-10, and IL-17A were increased in intestinal lymphocytes from E. acervulina-, E. maxima-, and/or E. tenella-infected chickens vaccinated with profilin/ISA 71 compared with profilin alone. None of these effects were seen in chickens injected with ISA 71 alone indicating that the adjuvant was not conferring non-specific immune stimulation. These results suggest that profilin plus ISA 71 augments protective immunity against selective Eimeria species in chickens.

Vaccination with Clostridium perfringens recombinant proteins in combination with Montanide™ ISA 71 VG adjuvant increases protection against experimental necrotic enteritis in commercial broiler chickens.

This study was performed to compare four Clostridium perfringens recombinant proteins as vaccine candidates using the Montanide™ ISA 71 VG adjuvant in an experimental model of necrotic enteritis. Broiler chickens were immunized subcutaneously with purified clostridial recombinant NetB toxin, pyruvate: ferredoxin oxidoreductase (PFO), α-toxin, or elongation factor-Tu (EF-Tu), or with vehicle control, in conjunction with ISA 71 VG, and intestinal lesion scores, body weight gains, NetB toxin and PFO antibody levels, and proinflammatory cytokine and chemokine levels were measured as outcomes of protection following oral co-infection with C. perfringens and Eimeria maxima. Birds immunized with all recombinant proteins plus ISA 71 VG showed significantly reduced gut lesions compared with the ISA 71 VG-only group. Birds immunized with NetB toxin or PFO plus ISA 71 VG exhibited significantly increased body weight gains compared with the ISA 71 VG alone group. Greater NetB toxin antibody titers were observed in the NetB/ISA 71 VG group, and greater PFO antibody titers were evident in the PFO/ISA 71 VG group, each compared with the other three vaccine/adjuvant groups. Finally, decreased levels of gene transcripts encoding interleukin-8, tumor necrosis factor superfamily 15, and LPS-induced TNF-α factor were observed in the intestinal lymphocytes of chickens immunized with NetB toxin, PFO, α-toxin, and/or EF-Tu in the presence of ISA 71 VG compared with ISA 71 VG alone. All parameters evaluated were equal in co-infected chickens given ISA 71 VG alone compared with infected/adjuvant-free birds, indicating that the adjuvant itself did not have a disease protective effect. These results suggest that vaccination with clostridial recombinant proteins, particularly NetB toxin or PFO, in combination with ISA 71 VG enhances protective immunity against experimental necrotic enteritis in broiler chickens.

Efficacy of intranasal and spray delivery of adjuvanted live vaccine against infectious bronchitis virus in experimentally infected poultry.

Live vaccines are widely used in the avian industry. Such vaccines can be either injected or delivered on animal mucosa and are usually not adjuvanted. In this study we show that live vaccines efficacy can be improved by formulation with adjuvants in a model of mucosal delivery of live infectious bronchitis vaccine in chicken. Three adjuvant technologies have been tested using intranasal and spray delivery methods to poultry. Those technologies are water in oil in water emulsion, nanoparticles and polymer adjuvants. Intranasal delivery of polymer and nanoparticles adjuvanted live vaccines improved significantly the antibody titer and protection to challenge observed compared to a commercial non-adjuvanted reference. Moreover, spray delivery of the polymer adjuvanted vaccine showed a significantly higher protection compared to the non-adjuvanted reference. Our data demonstrates that the use of MontanideTM adjuvants in the formulation of live poultry vaccines for mucosal delivery can confer to vaccinated animals a significantly improved protection against pathogens.

Load reduction in live PRRS vaccines using oil and polymer adjuvants.

PRRSV live vaccines are widely used in pig farming practice and are usually not adjuvanted. For safety issues, it would be useful to reduce the antigenic load of such vaccines while preserving their efficacy. In this study we show that the addition of polymer or oil adjuvants in a PRRS live vaccine enhanced the protection to challenge of vaccinated animals compared to a non-adjuvanted commercial reference. Moreover, for both types of adjuvants, despite lower antibody titers, the protection to challenge given by the adjuvanted vaccine containing only 50% of the antigen load was equivalent to the protection given by the non-adjuvanted vaccine. These results demonstrate that the addition of relevant adjuvants can enhance the efficacy of the protection conferred to animals by live vaccines.

Montanide IMS 1313 N VG PR nanoparticle adjuvant enhances antigen-specific immune responses to profilin following mucosal vaccination against Eimeria acervulina.

This study investigated protection against Eimeria acervulina (E. acervulina) following vaccination of chickens with an Eimeria recombinant profilin in conjunction with different adjuvants, or by changing the route of administration of the adjuvants. Day-old broilers were immunized twice with profilin emulsified in Montanide IMS 1313 N VG PR adjuvant (oral, nasal, or ocular routes), Montanide ISA 71 VG adjuvant (subcutaneous route), or Freund's adjuvant (subcutaneous route) and orally challenged with virulent E. acervulina parasites. Birds orally immunized with profilin plus IMS 1313 N VG PR, or subcutaneously immunized with profilin plus ISA 71 VG, had increased body weight gains compared with animals nasally or ocularly immunized with profilin plus IMS 1313 N VG PR, or subcutaneously immunized with profilin plus Freund's adjuvant. All adjuvant formulations, except for IMS 1313 N VG PR given by the nasal or ocular routes, decreased fecal parasite excretion and/or reduced intestinal lesions, compared with non-vaccinated and infected controls. Compared with animals vaccinated with profilin plus Freund's adjuvant, chickens immunized with profilin plus IMS 1313 N VG PR or ISA 71 VG showed higher post-infection intestinal levels of profilin-reactive IgY and secretary IgA antibodies. Finally, immunization with profilin in combination with ISA 71 VG was consistently better than profilin plus IMS 1313 N VG PR or Freund's adjuvant for increasing the percentages of CD4(+), CD8(+), BU1(+), TCR1(+), and TCR2(+) intestinal lymphocytes. These results indicate that experimental immunization of chickens with the recombinant profilin subunit vaccine in conjunction with IMS 1313 or ISA 71 VG adjuvants increases protective mucosal immunity against E. acervulina infection.

Mucosal immunity against Eimeria acervulina infection in broiler chickens following oral immunization with profilin in Montanide™ adjuvants.

The present study was conducted to compare aqueous nanoparticle-based Montanide™ IMS 1313 N VG PR (IMS 1313) and oil-based ISA 71 VG (ISA71) adjuvants in combination with an Eimeria subunit protein vaccine on protection against avian coccidiosis. Male broiler chicks were vaccinated twice with an Eimeria recombinant profilin protein alone or in conjunction with IMS 1313 or ISA 71 prior to infection with live, sporulated Eimeria acervulina oocysts. For comparison, chickens were immunized with a commercial live coccidiosis vaccine (Coccivac-B). The following parameters were assessed as measures of protective immunity: body weight gain, fecal oocyst output, profilin-specific intestinal secretary IgA (sIgA) or IgY antibody levels, and percentages of CD4(+), CD8(+), TCR1(+), or TCR2(+) intestinal intraepithelial lymphocytes (IELs). Birds vaccinated with profilin plus ISA 71 had increased body weight gains, equivalent to Coccivac-B vaccination, compared with the profilin-only group. Immunization with profilin plus IMS 1313, or with profilin plus ISA 71, reduced fecal oocysts shedding compared with the profilin-only group. Intestinal sIgA levels were greater in the profilin plus IMS 1313 or ISA 71 groups, and IgY levels were greater in the profilin plus ISA 71 group, compared with profilin alone. Birds vaccinated with profilin plus IMS 1313 or ISA 71 had higher percentages of CD4(+), CD8(+), and TCR1(+), but not TCR2(+), intestinal IELs compared with the profilin-only vaccinated group. Taken together, these results indicate that immunization of chickens with the recombinant profilin subunit vaccine in conjunction with IMS 1313 or ISA 71 adjuvants increases protective immunity against experimental E. acervulina infection.

Montanide™ ISA 71 VG adjuvant enhances antibody and cell-mediated immune responses to profilin subunit antigen vaccination and promotes protection against Eimeria acervulina and Eimeria tenella.

The present study was conducted to investigate the immunoenhancing effects of Montanide™ ISA 71 VG adjuvant on profilin subunit antigen vaccination. Broiler chickens were immunized subcutaneously with a purified Eimeria acervulina recombinant profilin protein, either alone or mixed with ISA 71 VG, and host immune responses were evaluated. After secondary immunization, antigen-specific antibody and T-cell responses were higher in the group which received profilin plus ISA 71 VG compared with the other groups. Furthermore, body weight gains and fecal oocyst shedding were evaluated following oral challenge infection with live E. acervulina or Eimeria tenella oocysts. Vaccination with profilin plus ISA 71 VG reduced oocyst shedding compared with animals immunized with profilin alone. These results demonstrate that the recombinant profilin subunit vaccine, when given in combination with Montanide™ ISA 71 VG, augments protective immunity against E. acervulina and E. tenella.

Immunoenhancing effects of Montanide ISA oil-based adjuvants on recombinant coccidia antigen vaccination against Eimeria acervulina infection.

The current study was conducted to investigate the immunoenhancing effects of Montanide adjuvants on protein subunit vaccination against avian coccidiosis. Broiler chickens were immunized subcutaneously with a purified Eimeria acervulina recombinant profilin protein, either alone or mixed with one of four adjuvants (ISA 70 VG, ISA 71 VG, ISA 201 VG or ISA 206 VG), and body weight gains, fecal oocyst shedding, and humoral and innate immune responses were evaluated following oral challenge infection with live E. acervulina oocysts. Immunization with profilin plus ISA 70 VG or ISA 71 VG increased body weight gains compared with vaccination with profilin alone. Profilin plus ISA 71 VG also reduced fecal oocyst shedding compared with vaccination in the absence of adjuvant. All adjuvants enhanced profilin serum antibody titers. Increased levels of gene transcripts encoding IL-2, IL-10, IL-17A, and IFN-gamma, but decreased levels of IL-15 mRNAs, were seen in intestinal intraepithelial lymphocytes of chickens immunized with profilin plus adjuvants compared with immunization with profilin alone. Finally, increased infiltration of lymphocytes, especially CD8(+) lymphocytes at the site of immunization was observed in birds given profilin plus ISA 71 VG compared with profilin alone. These results demonstrate that vaccination with the E. acervulina profilin subunit vaccine in combination with Montanide adjuvants enhances protective immunity against avian coccidiosis.

A new subunit vaccine based on nucleoprotein nanoparticles confers partial clinical and virological protection in calves against bovine respiratory syncytial virus.

Human and bovine respiratory syncytial viruses (HRSV and BRSV) are two closely related, worldwide prevalent viruses that are the leading cause of severe airway disease in children and calves, respectively. Efficacy of commercial bovine vaccines needs improvement and no human vaccine is licensed yet. We reported that nasal vaccination with the HRSV nucleoprotein produced as recombinant ring-shaped nanoparticles (N(SRS)) protects mice against a viral challenge with HRSV. The aim of this work was to evaluate this new vaccine that uses a conserved viral antigen, in calves, natural hosts for BRSV. Calves, free of colostral or natural anti-BRSV antibodies, were vaccinated with N(SRS) either intramuscularly, or both intramuscularly and intranasally using Montanide ISA71 and IMS4132 as adjuvants and challenged with BRSV. All vaccinated calves developed anti-N antibodies in blood and nasal secretions and N-specific cellular immunity in local lymph nodes. Clinical monitoring post-challenge demonstrated moderate respiratory pathology with local lung tissue consolidations for the non-vaccinated calves that were significantly reduced in the vaccinated calves. Vaccinated calves had lower viral loads than the non-vaccinated control calves. Thus N(SRS) vaccination in calves provided cross-protective immunity against BRSV infection without adverse inflammatory reaction.

Influence of adjuvant formulation on the induced protection of mice immunized with total soluble antigen of Trichinella spiralis.

Vaccination of pigs against the helminth nematode Trichinella could be a good alternative to prevent the risk of human infection. In order to develop an efficient and safe vaccine, the choice of the adjuvant is an important issue. In this study, two adjuvants were selected to prepare vaccines based on total soluble Trichinella spiralis muscle larvae (ML) antigen: Montanide ISA 70 water in oil emulsion and Montanide IMS nanoparticles. Aluminium hydroxide was used as a reference adjuvant. The immune response was checked by ELISA of parasite antigen specific IgG1 and IgE. Finally, protection induced in vaccinated mice was measured after a T. spiralis challenge by counting ML burdens. The results clearly showed an impact of adjuvants on the specific IgG1 and IgE antibody responses against T. spiralis. Differences were observed between the rates of protection induced according to the type of formulation, although the three adjuvants tested were able to enhance the humoral immune response. This work demonstrated the need to use an adjuvant to obtain a specific IgG1 and IgE responses directed against the total soluble extract of T. spiralis.