Protective Cell-mediated Immune Responses Research Articles

A split influenza vaccine formulated with a combination adjuvant composed of alpha-d-glucan nanoparticles and a STING agonist elicits cross-protective immunity in pigs

BackgroundSwine influenza A viruses (SwIAVs) pose an economic and pandemic threat, and development of novel effective vaccines is of critical significance. We evaluated the performance of split swine influenza A virus (SwIAV) H1N2 antigens with a plant-derived nanoparticle adjuvant alone (Nano-11) [Nano11-SwIAV] or in combination with the synthetic stimulator of interferon genes (STING) agonist ADU-S100 (NanoS100-SwIAV). Specific pathogen free (SPF) pigs were vaccinated twice via intramuscular (IM) or intradermal (ID) routes and challenged with a virulent heterologous SwIAV H1N1-OH7 virus.ResultsAnimals vaccinated IM or ID with NanoS100-SwIAV had significantly increased cross-reactive IgG and IgA titers in serum, nasal secretion and bronchoalveolar lavage fluid at day post challenge 6 (DPC6). Furthermore, NanoS100-SwIAV ID vaccinates, even at half the vaccine dose compared to their IM vaccinated counterparts, had significantly increased frequencies of CXCL10+ myeloid cells in the tracheobronchial lymph nodes (TBLN), and IFNγ+ effector memory T-helper/memory cells, IL-17A+ total T-helper/memory cells, central and effector memory T-helper/memory cells, IL-17A+ total cytotoxic T-lymphocytes (CTLs), and early effector CTLs in blood compared with the Nano11-SwIAV group demonstrating a potential dose-sparing effect and induction of a strong IL-17A+ T-helper/memory (Th17) response in the periphery. However, the frequencies of IFNγ+ late effector CTLs and effector memory T-helper/memory cells, IL-17A+ total CTLs, late effector CTLs, and CXCL10+ myeloid cells in blood, as well as lung CXCL10+ plasmacytoid dendritic cells were increased in NanoS100-SwIAV IM vaccinated pigs. Increased expression of IL-4 and IL-6 mRNA was observed in TBLN of Nano-11 based IM vaccinates following challenge. Furthermore, the challenge virus load in the lungs and nasal passage was undetectable in NanoS100-SwIAV IM vaccinates by DPC6 along with reduced macroscopic lung lesions and significantly higher virus neutralization titers in lungs at DPC6. However, NanoS100-SwIAV ID vaccinates exhibited significant reduction of challenge virus titers in nasal passages and a remarkable reduction of challenge virus in lungs.ConclusionsDespite vast genetic difference (77% HA gene identity) between the H1N2 and H1N1 SwIAV, the NanoS100 adjuvanted vaccine elicited cross protective cell mediated immune responses, suggesting the potential role of this combination adjuvant in inducing cross-protective immunity in pigs.Graphical

What We Need to Consider During and After the SARS-CoV-2 Pandemic.

Even though extreme containment and mitigation strategies were implemented by numerous governments around the world to slow down the spread of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the number of critically ill patients and fatalities keeps rising. This crisis has highlighted the socioeconomic disparities of health care systems within and among countries. As new CoVID policies and responses are implemented to lessen the impact of the virus, it is imperative (1) to consider additional mitigation strategies critical for the development of effective countermeasures, (2) to promote long-term policies and strict regulations of the trade of wildlife and live animal markets, and (3) to advocate for necessary funding and investments in global health, specifically for the prevention of and response to natural and manmade pandemics. This document considers some of these challenges.

Salmonella Enteritidis ghost vaccine carrying the hemagglutinin globular head (HA1) domain from H1N1 virus protects against salmonellosis and influenza in chickens

This study evaluated the attenuated Salmonella Enteritidis (SE) ghost strain JOL2114 (Δlon ΔcpxR Δasd), which displays on the bacterial surface the H1N1 hemagglutinin globular head portion (HA1; amino acid residues 63-286) on the bacterial surface for protective efficacy against Salmonella and H1N1 challenge in the chicken model, as the birds are the predominant reservoirs for both diseases. The ghost system enhanced the lysis process by converging two lysis processes found in bacteriophages: bacteriophage PhiX174 lysis gene E and holin-endolysin genes found in bacteriophage λ, complemented with accessory lysis-related proteins Rz/Rz1. The present lysis machinery resulted in complete lysis of host-attenuated SE strains in about 24 hrs of incubation under a non-permissible temperature of 42 °C in the absence of L-arabinose, an antisense inducer that blocks lysis gene expression during the growth phase. SE ghost JOL2114 surface display of HA1 was confirmed by Western blot analysis resulting in an immune-reactive band of 31 kDa in size. Chicken immunization via intramuscular and oral routes yielded both SE and HA1 antigen-specific immune responses. Protective humoral and cell-mediated immune responses were effectively elicited against both Salmonella and influenza challenge. This efficient strategy of ghost generation employs a dual system of phage lysis for biological generation of SE ghosts that preserves the surface antigenic architecture, offering a rapid and effective way to generate vaccines that could be deployed in urgent circumstances to protect against both Salmonella and influenza infection.

Deletion of Two Genes in Burkholderia pseudomallei MSHR668 That Target Essential Amino Acids Protect Acutely Infected BALB/c Mice and Promote Long Term Survival.

Melioidosis is an emerging disease that is caused by the facultative intracellular pathogen Burkholderia pseudomallei. It is intrinsically resistant to many antibiotics and host risk factors play a major role in susceptibility to infection. Currently, there is no human or animal vaccine against melioidosis. In this study, multiple B. pseudomallei MSHR668 deletion mutants were evaluated as live attenuated vaccines in the sensitive BALB/c mouse model of melioidosis. The most efficacious vaccines after an intraperitoneal challenge with 50-fold over the 50% median lethal dose (MLD50) with B. pseudomallei K96243 were 668 ΔhisF and 668 ΔilvI. Both vaccines completely protected mice in the acute phase of infection and showed significant protection (50% survivors) during the chronic phase of infection. The spleens of the survivors that were examined were sterile. Splenocytes from mice vaccinated with 668 ΔhisF and 668 ΔilvI expressed higher amounts of IFN-γ after stimulation with B. pseudomallei antigens than splenocytes from mice vaccinated with less protective candidates. Finally, we demonstrate that 668 ΔhisF is nonlethal in immunocompromised NOD/SCID mice. Our results show that 668 ΔhisF and 668 ΔilvI provide protective cell-mediated immune responses in the acute phase of infection and promote long term survival in the sensitive BALB/c mouse model of melioidosis.

Protective effect of intranasal peste des petits ruminants virus and bacterin vaccinations: Clinical, hematological, serological, and serum oxidative stress changes in challenged goats.

Background and Aim:The current vaccination for peste des petits ruminants virus (PPRV) is stalled by myriad challenges and continuous endemicity of pneumonia due to fulminant bacterial complication in goats. The present study evaluated the protective effect of intranasal PPRV linage 1 and bacterine vaccinations.Materials and Methods:Twelve West African Dwarf (WAD) goats aged 6 months were randomly grouped and vaccinated within 2 weeks using a combination of PPRV lineage 1 vaccine (Nig/75), and bacterin from Mannheimia haemolytica (Mh) or Pasteurella multocida intranasally. The goats were observed for 3 weeks post-vaccination before comingled with a known infected WAD goat with apparent clinical signs of peste des petits ruminants and further observed clinically for 5 weeks post-infection (PI). Blood samples were taken for hematology while sera were assayed for antioxidants (glutathione peroxidase, glutathione transferase, and superoxide dismutase) activities and pro-oxidants (malondialdehyde content, reduced glutathione, hydrogen peroxide generation, and myeloperoxidase) using spectrophotometric methods. Data were subjected to parametric statistics at α=0.05 using GraphPad Prism version 21.Results:Clinically, there were pyrexia, oculonasal discharge, diarrhea, anemia, leukopenia, and increased pro-oxidants in the unvaccinated goats, while moderate neutrophilia and leukocytosis were observed in PPRV and bacterin vaccinated goats. Two unvaccinated goats were weak and euthanized at 13 and 28 days PI. The goats vaccinated with PPRV and Mh showed better response clinically and biochemically.Conclusion:The mucosal vaccination of goats with PPRV vaccine and bacterine will protect against exposure and culminate in the development of protective mucosal, humoral, and cell-mediated immune responses. This vaccination strategy will provide framework needed in the prevention and control of endemic caprine pneumonia in Nigeria.

Intranasal inoculate of influenza virus vaccine against lethal virus challenge

Vaccine adjuvants are essential for enhancing immune responses during vaccination. However, only a limited number of safe and effective adjuvants, especially mucosal adjuvants, are available for use in vaccines. The development of a practically applicable mucosal adjuvant is therefore urgently needed. Here, we showed that the non-toxic CTA1-DD adjuvant, which combined the full enzymatic activity of the A1 subunit of cholera toxin (CT) with two immunoglobulin-binding domains of Staphylococcus aureus protein A (SpA), promoted mucosal and systemic humoral and cell-mediated immune responses following intranasal administration with H1N1 split vaccine in mice. We demonstrated that CTA1-DD-adjuvant vaccine provided 100% protection against mortality and greatly reduced morbidity in a mouse model. We also showed that addition of CTA1-DD to the vaccine elicited significantly higher hemagglutination inhibition titers and IgG antibodies in sera than alum adjuvant. Furthermore, CTA1-DD significantly promoted the production of mucosal secretory IgA in lung lavages and vaginal lavages. We also showed that CTA1-DD could be used as a mucosal adjuvant to enhance T cell responses. Our results clearly indicated that CTA1-DD contributed to the elicitation of a protective cell-mediated immune response required for efficacious vaccination against influenza virus, which suggested that this adjuvant could be explored further as a clinically safe mucosal vaccine adjuvant for respiratory diseases and other mucosal diseases.

A strong adjuvant based on glycol-chitosan-coated lipid-polymer hybrid nanoparticles potentiates mucosal immune responses against the recombinant Chlamydia trachomatis fusion antigen CTH522

Induction of mucosal immunity with vaccines is attractive for the immunological protection against pathogen entry directly at the site of infection. An example is infection with Chlamydia trachomatis (Ct), which is the most common sexually transmitted infection in the world, and there is an unmet medical need for an effective vaccine. A vaccine against Ct should elicit protective humoral and cell-mediated immune (CMI) responses in the genital tract mucosa. We previously designed an antibody- and CMI-inducing adjuvant based on poly(dl-lactic-co-glycolic acid) (PLGA) nanoparticles modified with the cationic surfactant dimethyldioctadecylammonium bromide and the immunopotentiator trehalose-6,6′-dibehenate. Here we show that immunization with these lipid-polymer hybrid nanoparticles (LPNs) coated with the mucoadhesive polymer chitosan enhances mucosal immune responses. Glycol chitosan (GC)-modified LPNs were engineered using an oil-in-water single emulsion solvent evaporation method. The nanoparticle design was optimized in a highly systematic way by using a quality-by-design approach to define the optimal operating space and to gain maximal mechanistic information about the GC coating of the LPNs. Cryo-transmission electron microscopy revealed a PLGA core coated with one or several concentric lipid bilayers. The GC coating of the surface was identified as a saturable, GC concentration-dependent increase in particle size and a reduction of the zeta-potential, and the coating layer could be compressed upon addition of salt. Increased antigen-specific mucosal immune responses were induced in the lungs and the genital tract with the optimized GC-coated LPN adjuvant upon nasal immunization of mice with the recombinant Ct fusion antigen CTH522. The mucosal responses were characterized by CTH522-specific IgG/IgA antibodies, together with CTH522-specific interferon γ-producing Th1 cells. This study demonstrates that mucosal administration of CTH522 adjuvanted with chitosan-coated LPNs represents a promising strategy to modulate the magnitude of mucosal vaccine responses.

Mucosal Administration of Cycle-Di-Nucleotide-Adjuvanted Virosomes Efficiently Induces Protection against Influenza H5N1 in Mice.

The need for more effective influenza vaccines is highlighted by the emergence of novel influenza strains, which can lead to new pandemics. There is a growing population of susceptible subjects at risk for severe complications of influenza, such as the elderly who are only in part protected by current licensed seasonal vaccines. One strategy for improving seasonal and pandemic vaccines takes advantage of adjuvants to boost and modulate evoked immune responses. In this study, we examined the capacity of the recently described adjuvant cyclic di-adenosine monophosphate (c-di-AMP) to serve as an adjuvant for improved mucosal influenza vaccines, and induce effective protection against influenza H5N1. In detail, c-di-AMP promoted (i) effective local and systemic humoral immune responses, including protective hemagglutination inhibition titers, (ii) effective cellular responses, including multifunctional T cell activity, (iii) induction of long-lasting immunity, and (iv) protection against viral challenge. Furthermore, we demonstrated the dose-sparing capacity of the adjuvant as well as the ability to evoke cross-clade protective immune responses. Overall, our results suggest that c-di-AMP contributes to the generation of a protective cell-mediated immune response required for efficacious vaccination against influenza, which supports the further development of c-di-AMP as an adjuvant for seasonal and pandemic influenza mucosal vaccines.

In silico analysis and in vitro evaluation of immunogenic and immunomodulatory properties of promiscuous peptides derived from Leishmania infantum eukaryotic initiation factor

It is generally considered as imperative the ability to control leishmaniasis through the development of a protective vaccine capable of inducing long-lasting and protective cell-mediated immune responses. In this current study, we demonstrated potential epitopes that bind to H2 MHC class I and II molecules by conducting the in silico analysis of Leishmania infantum eukaryotic Initiation Factor (LieIF) protein, using online available algorithms. Moreover, we synthesized five peptides (16–18 amino acids long) which are part of the N-terminal portion of LieIF and contain promising MHC class I and II-restricted epitopes and afterwards, their predicted immunogenicity was evaluated in vitro by monitoring peptide-specific T-cell responses. Additionally, the immunomodulatory properties of these peptides were investigated in vitro by exploring their potential of inducing phenotypic maturation and functional differentiation of murine Bone-Marrow derived Dendritic Cells (BM-DCs). It was revealed by our data that all the synthetic peptides predicted for H2 alleles; present the property of immunogenicity. Among the synthetic peptides which contained T-cell epitopes, the peptide 52–68 aa (LieIF_2) exhibited immunomodulatory properties with the larger potential. LieIF_2-pulsed BM-DCs up-regulated the expression of the co-stimulatory surface molecules CD80 and CD86, as well as the production of the proinflammatory cytokine TNF-α and of the Th1-polarizing cytokines IL-12 and IFN-γ. The aforementioned data suggest that selected parts of LieIF could be used to develop innovative subunit protective vaccines able to induce effective immunity mediated by MHC class I-restricted as well as class II-restricted T-cell responses.

Interleukin 10 inhibits pro-inflammatory cytokine responses and killing of Burkholderia pseudomallei

Melioidosis, caused by Burkholderia pseudomallei, is endemic in northeastern Thailand and Northern Australia. Severe septicemic melioidosis is associated with high levels of pro-inflammatory cytokines and is correlated with poor clinical outcomes. IL-10 is an immunoregulatory cytokine, which in other infections can control the expression of pro-inflammatory cytokines, but its role in melioidosis has not been addressed. Here, whole blood of healthy seropositive individuals (n = 75), living in N. E. Thailand was co-cultured with B. pseudomallei and production of IL-10 and IFN-γ detected and the cellular sources identified. CD3− CD14+ monocytes were the main source of IL-10. Neutralization of IL-10 increased IFN-γ, IL-6 and TNF-α production and improved bacteria killing. IFN-γ production and microbicidal activity were impaired in individuals with diabetes mellitus (DM). In contrast, IL-10 production was unimpaired in individuals with DM, resulting in an IL-10 dominant cytokine balance. Neutralization of IL-10 restored the IFN-γ response of individuals with DM to similar levels observed in healthy individuals and improved killing of B. pseudomallei in vitro. These results demonstrate that monocyte derived IL-10 acts to inhibit potentially protective cell mediated immune responses against B. pseudomallei, but may also moderate the pathological effects of excessive cytokine production during sepsis.

Immunization with Attenuated Equine Herpesvirus 1 Strain KyA Induces Innate Immune Responses That Protect Mice from Lethal Challenge.

Equine herpesvirus 1 (EHV-1) is a major pathogen affecting equines worldwide. The virus causes respiratory disease, abortion, and, in some cases, neurological disease. EHV-1 strain KyA is attenuated in the mouse and equine, whereas wild-type strain RacL11 induces severe inflammation of the lung, causing infected mice to succumb at 4 to 6 days postinfection. Our previous results showed that KyA immunization protected CBA mice from pathogenic RacL11 challenge at 2 and 4 weeks postimmunization and that KyA infection elicited protective humoral and cell-mediated immune responses. To investigate the protective mechanisms of innate immune responses to KyA, KyA-immunized mice were challenged with RacL11 at various times postvaccination. KyA immunization protected mice from RacL11 challenge at 1 to 7 days postimmunization. Immunized mice lost less than 10% of their body weight and rapidly regained weight. Virus titers in the lungs of KyA-immunized mice were 1,000-fold lower at 2 days post-RacL11 challenge than virus titers in the lungs of nonimmunized mice, indicating accelerated virus clearance. Affymetrix microarray analysis revealed that gamma interferon (IFN-γ) and 16 antiviral interferon-stimulated genes (ISGs) were upregulated 3.1- to 48.2-fold at 8 h postchallenge in the lungs of RacL11-challenged mice that had been immunized with KyA. Murine IFN-γ inhibited EHV-1 infection of murine alveolar macrophages and protected mice against lethal EHV-1 challenge, suggesting that IFN-γ expression is important in mediating the protection elicited by KyA immunization. These results suggest that EHV-1 KyA may be used as a live attenuated EHV-1 vaccine as well as a prophylactic agent in horses. Viral infection of cells initiates a signal cascade of events that ultimately attempts to limit viral replication and prevent infection through the expression of host antiviral proteins. In this study, we show that EHV-1 KyA immunization effectively protected CBA mice from pathogenic RacL11 challenge at 1 to 7 days postvaccination and increased the expression of IFN-γ and 16 antiviral interferon-stimulated genes (ISGs). The administration of IFN-γ blocked EHV-1 replication in murine alveolar macrophages and mouse lungs and protected mice from lethal challenge. To our knowledge, this is the first report of an attenuated EHV-1 vaccine that protects the animal at 1 to 7 days postimmunization by innate immune responses. Our findings suggested that IFN-γ serves as a novel prophylactic agent and may offer new strategies for the development of anti-EHV-1 agents in the equine.

Protein vaccination with HPV16 E7/Pep-1 nanoparticles elicits a protective T-helper cell-mediated immune response.

Two human papillomavirus (HPV) viral oncoproteins, E6 and E7 represent ideal targets for development of a therapeutic HPV vaccine. It is important to reduce the rate of HPV-associated malignancies through improvement of vaccine modalities. In this study, we used a short amphipathic peptide carrier, Pep-1, for delivery of the full-length HPV16 E7 protein into mammalian cells and evaluated immune responses and protective effects of different formulations in C57BL/6 tumor mice model. Our results showed that the complexes of E7/Pep-1 protein form stable nanoparticles through noncovalent binding with an average size of 120 to 250 nm. The efficient delivery of E7 protein by Pep-1 at molar ratio of 1:20 was detected in HEK-293T cell line for 1 h and 3 h post-transfection. Immunization with E7/Pep-1 nanoparticles at a ratio of 1:20 induced a higher Th1 cellular immune response with the predominant IgG2a and IFN-γ levels than those induced by E7 protein in a murine tumor model. These data suggest that Pep-1 peptide would indicate promising applications for improvement of HPV therapeutic vaccines. © 2016 IUBMB Life, 68(6):459-467, 2016.

A therapeutic nanoparticle vaccine against Trypanosoma cruzi in a BALB/c mouse model of Chagas disease

ABSTRACTChagas disease, caused by Trypanosoma cruzi, results in an acute febrile illness that progresses to chronic chagasic cardiomyopathy in 30% of patients. Current treatments have significant side effects and poor efficacy during the chronic phase; therefore, there is an urgent need for new treatment modalities. A robust TH1-mediated immune response correlates with favorable clinical outcomes. A therapeutic vaccine administered to infected individuals could bolster the immune response, thereby slowing or stopping the progression of chagasic cardiomyopathy. Prior work in mice has identified an efficacious T. cruzi DNA vaccine encoding Tc24. To elicit a similar protective cell-mediated immune response to a Tc24 recombinant protein, we utilized a poly(lactic-co-glycolic acid) nanoparticle delivery system in conjunction with CpG motif-containing oligodeoxynucleotides as an immunomodulatory adjuvant. In a BALB/c mouse model, the vaccine produced a TH1-biased immune response, as demonstrated by a significant increase in antigen-specific IFNγ-producing splenocytes, IgG2a titers, and proliferative capacity of CD8+ T cells. When tested for therapeutic efficacy, significantly reduced systemic parasitemia was seen during peak parasitemia. Additionally, there was a significant reduction in cardiac parasite burden and inflammatory cell infiltrate. This is the first study demonstrating immunogenicity and efficacy of a therapeutic Chagas vaccine using a nanoparticle delivery system.

Predicting the Role of IL-10 in the Regulation of the Adaptive Immune Responses in Mycobacterium avium Subsp. paratuberculosis Infections Using Mathematical Models.

Mycobacterium avium subsp. paratuberculosis (MAP) is an intracellular bacterial pathogen that causes Johne’s disease (JD) in cattle and other animals. The hallmark of MAP infection in the early stages is a strong protective cell-mediated immune response (Th1-type), characterized by antigen-specific γ-interferon (IFN-γ). The Th1 response wanes with disease progression and is supplanted by a non-protective humoral immune response (Th2-type). Interleukin-10 (IL-10) is believed to play a critical role in the regulation of host immune responses to MAP infection and potentially orchestrate the reversal of Th1/Th2 immune dominance during disease progression. However, how its role correlates with MAP infection remains to be completely deciphered. We developed mathematical models to explain probable mechanisms for IL-10 involvement in MAP infection. We tested our models with IL-4, IL-10, IFN-γ, and MAP fecal shedding data collected from calves that were experimentally infected and followed over a period of 360 days in the study of Stabel and Robbe-Austerman (2011). Our models predicted that IL-10 can have different roles during MAP infection, (i) it can suppress the Th1 expression, (ii) can enhance Th2 (IL-4) expression, and (iii) can suppress the Th1 expression in synergy with IL-4. In these predicted roles, suppression of Th1 responses was correlated with increased number of MAP. We also predicted that Th1-mediated responses (IFN-γ) can lead to high expression of IL-10 and that infection burden regulates Th2 suppression by the Th1 response. Our models highlight areas where more experimental data is required to refine our model assumptions, and further test and investigate the role of IL-10 in MAP infection.

Programming of Influenza Vaccine Broadness and Persistence by Mucoadhesive Polymer-Based Adjuvant Systems.

The development of an anti-influenza vaccine with the potential for cross-protection against seasonal drift variants as well as occasionally emerging reassortant viruses is essential. In this study, we successfully generated a novel anti-influenza vaccine system combining conserved matrix protein 2 (sM2) and stalk domain of hemagglutinin (HA2) fusion protein (sM2HA2) and poly-γ-glutamic acid (γ-PGA)-based vaccine adjuvant systems that can act as a mucoadhesive delivery vehicle of sM2HA2 as well as a robust strategy for the incorporation of hydrophobic immunostimulatory 3-O-desacyl-4'-monophosphoryl lipid A (MPL) and QS21. Intranasal coadministration of sM2HA2 and the combination adjuvant γ-PGA/MPL/QS21 (CA-PMQ) was able to induce a high degree of protective mucosal, systemic, and cell-mediated immune responses. The sM2HA2/CA-PMQ immunization was able to prevent disease symptoms, confering complete protection against lethal infection with divergent influenza subtypes (H5N1, H1N1, H5N2, H7N3, and H9N2) that lasted for at least 6 mo. Therefore, our data suggest that mucosal administration of sM2HA2 in combination with CA-PMQ could be a potent strategy for a broad cross-protective influenza vaccine, and CA-PMQ as a mucosal adjuvant could be used for effective mucosal vaccines.

Maintenance of brucellosis in Yellowstone bison: linking seasonal food resources, host-pathogen interaction, and life-history trade-offs.

The seasonal availability of food resources is an important factor shaping the life-history strategies of organisms. During times of nutritional restriction, physiological trade-offs can induce periods of immune suppression, thereby increasing susceptibility to infectious disease. Our goal was to provide a conceptual framework describing how the endemic level bovine brucellosis (Brucella abortus) may be maintained in Yellowstone bison based on the seasonality of food resources and the life-history strategies of the host and pathogen. Our analysis was based on active B. abortus infection (measured via bacterial culture), nutritional indicators (measured as metabolites and hormones in plasma), and carcass measurements of 402 slaughtered bison. Data from Yellowstone bison were used to investigate (1) whether seasonal changes in diet quality affect nutritional condition and coincide with the reproductive needs of female bison; (2) whether active B. abortus infection and infection intensities vary with host nutrition and nutritional condition; and (3) the evidence for seasonal changes in immune responses, which may offer protection against B. abortus, in relation to nutritional condition. Female bison experienced a decline in nutritional condition during winter as reproductive demands of late gestation increased while forage quality and availability declined. Active B. abortus infection was negatively associated with bison age and nutritional condition, with the intensity of infection negatively associated with indicators of nutrition (e.g., dietary protein and energy) and body weight. Data suggest that protective cell-mediated immune responses may be reduced during the B. abortus transmission period, which coincides with nutritional insufficiencies and elevated reproductive demands during spring. Our results illustrate how seasonal food restriction can drive physiological trade-offs that suppress immune function and create infection and transmission opportunities for pathogens.

Choice and Design of Adjuvants for Parenteral and Mucosal Vaccines.

The existence of pathogens that escape recognition by specific vaccines, the need to improve existing vaccines and the increased availability of therapeutic (non-infectious disease) vaccines necessitate the rational development of novel vaccine concepts based on the induction of protective cell-mediated immune responses. For naive T-cell activation, several signals resulting from innate and adaptive interactions need to be integrated, and adjuvants may interfere with some or all of these signals. Adjuvants, for example, are used to promote the immunogenicity of antigens in vaccines, by inducing a pro-inflammatory environment that enables the recruitment and promotion of the infiltration of phagocytic cells, particularly antigen-presenting cells (APC), to the injection site. Adjuvants can enhance antigen presentation, induce cytokine expression, activate APC and modulate more downstream adaptive immune reactions (vaccine delivery systems, facilitating immune Signal 1). In addition, adjuvants can act as immunopotentiators (facilitating Signals 2 and 3) exhibiting immune stimulatory effects during antigen presentation by inducing the expression of co-stimulatory molecules on APC. Together, these signals determine the strength of activation of specific T-cells, thereby also influencing the quality of the downstream T helper cytokine profiles and the differentiation of antigen-specific T helper populations (Signal 3). New adjuvants should also target specific (innate) immune cells in order to facilitate proper activation of downstream adaptive immune responses and homing (Signal 4). It is desirable that these adjuvants should be able to exert such responses in the context of mucosal administered vaccines. This review focuses on the understanding of the potential working mechanisms of the most well-known classes of adjuvants to be used effectively in vaccines.

Targeted Immunology for Prevention and Cure of VL

Leishmaniasis is a neglected tropical disease caused by a group of protozoan parasites of the genus Leishmania. Clinical presentation of leishmaniasis can range from cutaneous, mucocutaneous, or visceral forms depending on the parasite species. Visceral leishmaniasis (VL) caused by L. donovani and L. infantum is the severest and one of the deadliest parasitic diseases of the tropics second only to malaria (1). Nearly, 20,000‐40,000 annual deaths are estimated due to this disease (2). Except for the Indian subcontinent and West Africa, VL is frequent in dogs, which serve as the major reservoir for zoonosis (3). Transmitted by the bite of an infected sandfly, Leishmania endure in the phagolysomal compartment of macrophages by evasion and attenuation of the microbicidal functions of the host (4, 5). Leishmania has evolved as a successful parasite chiefly by its ability to modulate the immunological and cyto-chemical responses of the host following infection. The key strategy for successful pathogenesis is to subvert the nitric oxide burst in the host macrophage. This opportunistic parasite thus establishes a safe niche in the inactivated phagocyte and uncontrolled parasitization in liver, spleen, and bone marrow leads to symptomatic VL characterized by fever, weight loss, hepatosplenomegaly, and anemia (3). Since the pathogenesis of the disease is based on subversion and modulation of both innate and adaptive arms of immunity, the disease is opportunistic to immuno-suppression (6). Hence, commencement of an appropriate immune response is a challenge for the control of VL infection. Indeed, therapeutic drugs like SSG and miltefosine are immuno-modulators that trigger Th1 responses essential for activation of oxidative burst in the macrophages (7, 8). However, major limitations of narrow therapeutic index and increasing incidence of resistance with currently used drugs for VL are encumbrances in effective disease management. Recent approaches like combination therapy, targeted delivery, and use of immune-adjunct are efforts to bring down the effective doses of these toxicity-associated drugs. Most promising are the prospects of various immune-targeted therapeutic approaches for treatment of VL (9). Various leishmanial antigens, cytokines, and antibodies that initiate protective Th1-biased cell-mediated immune responses used singly or as an adjunct to conventional chemotherapy are potent immuno-chemotherapeutic agents for the cure of VL (10, 11). Additionally, medicinal plants and their products have opened new dimensions in search of safer and cheaper anti-leishmanial immuno-modulators. These phytochemicals are not only promising as immuno-chemotherapeutic agents against VL but also have potential as immuno-adjuvants and adjuncts to chemotherapy for a number of other immuno-regulatory diseases (12). Since both cure and resilience to Leishmania infection depend on the immunological status of the host, the antigens that can trigger healing responses can also induce prophylactic immunity. Therefore, identification of immunogens that can induce Th1 responses is the critical aspect of vaccine search against VL (13). Although a number of defined antigens have been reported to impart protective immunity against experimental VL, recent trend of reverse vaccinology is a promising aspect for identification of key immunogens for a successful vaccine (13, 14). This requires rational inputs and algorithm for identification of a promising antigen from the whole proteome data analysis in silico (15). One of the key inputs is to identify the epitopes for activation of both CD4 C Th1 and CD8 C T cells.

Attenuation and immunogenicity of host-range extended modified vaccinia virus Ankara recombinants

Modified vaccinia virus Ankara (MVA) is being widely investigated as a safe smallpox vaccine and as an expression vector to produce vaccines against other infectious diseases and cancer. MVA was isolated following more than 500 passages in chick embryo fibroblasts and suffered several major deletions and numerous small mutations resulting in replication defects in human and most other mammalian cells as well as severe attenuation of pathogenicity. Due to the host range restriction, primary chick embryo fibroblasts are routinely used for production of MVA-based vaccines. While a replication defect undoubtedly contributes to safety of MVA, it is worth considering whether host range and attenuation are partially separable properties. Marker rescue transfection experiments resulted in the creation of recombinant MVAs with extended mammalian cell host range. Here, we characterize two host-range extended rMVAs and show that they (i) have acquired the ability to stably replicate in Vero cells, which are frequently used as a cell substrate for vaccine manufacture, (ii) are severely attenuated in immunocompetent and immunodeficient mouse strains following intranasal infection, (iii) are more pathogenic than MVA but less pathogenic than the ACAM2000 vaccine strain at high intracranial doses, (iv) do not form lesions upon tail scratch in mice in contrast to ACAM2000 and (v) induce protective humoral and cell-mediated immune responses similar to MVA. The extended host range of rMVAs may be useful for vaccine production.

Topical CpG adjuvant enhances immune response to subcutaneous antigen by modulating the cutaneous lymph node environment (P4267)

Abstract Current vaccines are mainly administered by intramuscular route but the skin harbors many immune cells and can be used as a site of immunization. Topical CpG oligodeoxynucleotide (ODN), a Toll-like receptor 9 (TLR9) agonist, improves humoral and cell-mediated immune responses to locally injected protein-based vaccines in mice but the mechanisms are still unclear. Topical CpG ODN is internalized by CD11c+ cells that are detected within the skin draining lymph nodes (SLN) at 48 hours post treatment. Ablation of TLR9 in the hematopoietic compartment of bone marrow chimeric mice abrogates antigen-specific CD8+ T cells production. TLR9 ablation in the stromal compartment also decreases topical CpG adjuvant effect. Topical CpG ODN differentially modulates the environment of SLN compared to subcutaneous administration within 24 hours of treatment (with 4-7 fold increased expression of 4 genes: Ccl4, Cxcl3, Ifng and Il11). A higher proportion of antigen-specific CD4+ T cells in the SLN express tissue-homing molecules (P- and E-selectin ligand) when adjuvant is administered topically compared to subcutaneously. Egress of lymphocytes from SLN is necessary for optimal CpG adjuvant effect using a contact hypersensitivity model. We propose that topical CpG ODN activates stromal cells as well as hematopoietic-derived cells in the skin, to subsequently modulate the environment of SLN. These changes enhance protective humoral and cell-mediated immune responses.