Strong Cell-mediated Immune Responses Research Articles

The Challenges of Vaccine Development Against Betacoronaviruses: Antibody Dependent Enhancement and Sendai Virus as a Possible Vaccine Vector

To design an effective and safe vaccine against betacoronaviruses, it is necessary to elicit a combination of strong humoral and cell-mediated immune responses as well as to minimize the risk of antibody-dependent enhancement of viral infection. This phenomenon was observed in animal trials of experimental vaccines against SARS-CoV-1 and MERS-CoV that were developed based on inactivated coronavirus or vector constructs expressing the spike protein (S) of the virion. The substitution and glycosylation of certain amino acids in the antigenic determinants of the S-protein, as well as its conformational changes, can lead to the same effect in a new experimental vaccine against SARS-CoV-2. This review outlines approaches for developing vaccines against the new SARS-CoV-2 coronavirus that are based on non-pathogenic viral vectors. For efficient prevention of infections caused by respiratory pathogens the ability of the vaccine to stimulate mucosal immunity in the respiratory tract is important. Such a vaccine can be developed using non-pathogenic Sendai virus vector, since it can be administered intranasally and induce a mucosal immune response that strengthens the antiviral barrier in the respiratory tract and provides reliable protection against infection. The mucosal immunity and the production of IgA antibodies accompanying its development reduces the likelihood of developing an antibody-dependent infection enhancement, which is usually associated only with immunopathological IgG antibodies.

Vaccination with a live-attenuated small-colony variant improves the humoral and cell-mediated responses againstStaphylococcus aureus.

Staphylococcus aureus is known to produce persistent and chronic infections in both humans and animals. It is recognized that small-colony variants (SCVs), which produce higher levels of biofilm and that are capable of intracellular persistence, contribute to the chronicity or recurrence of infections and that this phenotype is inherent to the pathogenesis process. Prevention of S. aureus infections through vaccination has not yet met with considerable success. Some of the current vaccine formulations for S. aureus bovine mastitis consist of inactivated S. aureus bacteria, sometimes combined to E. coli J5. As such, the stimulation of cell-mediated immunity by these vaccines might not be optimal. With this in mind, we recently engineered a genetically stable double mutant SCV (ΔvraGΔhemB), which was highly attenuated in a mastitis model of infection. The present work describes the immune responses elicited in mice by various experimental vaccine compositions including the live-attenuated SCV double mutant and its inactivated form, combined or not with inactivated E. coli J5. The live-attenuated SCV was found to provoke a strong and balanced humoral response in immunized mice, as well as strong proliferation of ex-vivo stimulated splenocytes isolated from these animals. These splenocytes were also found to release high concentration of IL-17 and IFN-γ when compared to every other vaccination formulation. Inversely, the inactivated whole-cell vaccine, alone or in combination with the E. coli J5 bacterin, elicited lower antibody titers and failed to induce Th1 or Th17 cell-mediated responses in the splenocyte proliferation assay. Our results suggest that live-attenuated SCVs can trigger host immunity differently than inactivated bacteria and could represent a suitable vector for inducing strong humoral and cell-mediated immune responses, which are crucial for protection. This could represent an important improvement over existing vaccine formulations for preventing S. aureus bovine mastitis and other infections caused by this pathogen.

Simplified Admix Archaeal Glycolipid Adjuvanted Vaccine and Checkpoint Inhibitor Therapy Combination Enhances Protection from Murine Melanoma.

Archaeosomes are liposomes composed of natural or synthetic archaeal lipids that when used as adjuvants induce strong long-lasting humoral and cell-mediated immune responses against entrapped antigens. However, traditional entrapped archaeosome formulations have only low entrapment efficiency, therefore we have developed a novel admixed formulation which offers many advantages, including reduced loss of antigen, consistency of batch-to-batch production as well as providing the option to formulate the vaccine immediately before use, which is beneficial for next generation cancer therapy platforms that include patient specific neo-antigens or for use with antigens that are less stable. Herein, we demonstrate that, when used in combination with anti-CTLA-4 and anti-PD-1 checkpoint therapy, this novel admixed archaeosome formulation, comprised of preformed sulfated lactosyl archaeol (SLA) archaeosomes admixed with OVA antigen (SLA–OVA (adm)), was as effective at inducing strong CD8+ T cell responses and protection from a B16-OVA melanoma tumor challenge as the traditionally formulated archaeosomes with encapsulated OVA protein. Furthermore, archaeosome vaccine formulations combined with anti-CTLA-4 and anti-PD-1 therapy, induced OVA-CD8+ T cells within the tumor and immunohistochemical analysis revealed the presence of CD8+ T cells associated with dying or dead tumor cells as well as within or around tumor blood vessels. Overall, archaeosomes constitute an attractive option for use with combinatorial checkpoint inhibitor cancer therapy platforms.

Adenoviral vector-based vaccine is fully protective against lethal Lassa fever challenge in Hartley guinea pigs

Lassa virus (LASV), the causative agent of Lassa fever (LF), was first identified in 1969. Since then, outbreaks in the endemic countries of Nigeria, Liberia, and Sierra Leone occur on an annual basis resulting in a case-fatality rate of 15–70% in hospitalized patients. There is currently no licensed vaccine and there are limited animal models to test vaccine efficacy. An estimated 37.7 million people are at risk of contracting LASV; therefore, there is an urgent need for the development of a safe, effective vaccine against LASV infection. The LF endemic countries are also inflicted with HIV, Ebola, and malaria infections. The safety in immunocompromised populations must be considered in LASV vaccine development. The novel adenovirus vector-based platform, Ad5 (E1-,E2b-) has been used in clinical trial protocols for treatment of immunocompromised individuals, has been shown to exhibit high stability, low safety risk in humans, and induces a strong cell-mediated and pro-inflammatory immune response even in the presence of pre-existing adenovirus immunity. To this nature, our lab has developed an Ad5 (E1-,E2b-) vector-based vaccine expressing the LASV-NP or LASV-GPC. We found that guinea pigs vaccinated with two doses of Ad5 (E1-,E2b-) LASV-NP and Ad5 (E1-,E2b-) LASV-GPC were protected against lethal LASV challenge. The Ad5 (E1-,E2b-) LASV-NP and LASV-GPC vaccine represents a potential vaccine candidate against LF.

The immunomodulatory functions and molecular mechanism of a new bursal heptapeptide (BP7) in immune responses and immature B cells

The bursa of Fabricius (BF) is the acknowledged central humoural immune organ unique to birds and plays a vital role in B lymphocyte development. In addition, the unique molecular immune features of bursal-derived biological peptides involved in B cell development are rarely reported. In this paper, a novel bursal heptapeptide (BP7) with the sequence GGCDGAA was isolated from the BF and was shown to enhance the monoclonal antibody production of a hybridoma. A mouse immunization experiment showed that mice immunized with an AIV antigen and BP7 produced strong antibody responses and cell-mediated immune responses. Additionally, BP7 stimulated increased mRNA levels of sIgM in immature mouse WEHI-231 B cells. Gene microarray results confirmed that BP7 regulated 2465 differentially expressed genes in BP7-treated WEHI-231 cells and induced 13 signalling pathways and various immune-related functional processes. Furthermore, we found that BP7 stimulated WEHI-231 cell autophagy and AMPK-ULK1 phosphorylation and regulated Bcl-2 protein expression. Finally, chicken immunization showed that BP7 enhanced the potential antibody and cytokine responses to the AIV antigen. These results suggested that BP7 might be an active biological factor that functions as a potential immunopotentiator, which provided some novel insights into the molecular mechanisms of the effects of bursal peptides on immune functions and B cell differentiation.

Intragastric delivery of recombinant Lactococcus lactis displaying ectodomain of influenza matrix protein 2 (M2e) and neuraminidase (NA) induced focused mucosal and systemic immune responses in chickens

Compounding with the problem of frequent antigenic shift and occasional drift of the segmented genome of Avian Influenza Virus (AIV), vaccines based on major surface glycoproteins such as haemagglutinin (HA) to counter heterosubtypic AIV infection in chickens remain unsuccessful. In contrast, neuraminidase (NA), the second most abundant surface glycoprotein present in viral capsid is less mutable and, in some instances, successful in eliciting inter-species cross-reactive antibody responses. However, without selective activation of B-cells and T-cells, the ability of NA to induce strong cell mediated immune responses is limited, thus NA based vaccines cannot singularly address the risk of virus escape from host defence. To this end, the highly conserved ectodomain of influenza matrix protein-2 (M2e) has emerged as an attractive cross-protective vaccine target. The present study describes the potential of recombinant Lactococcus lactis (rL. lactis) in expressing functional influenza NA or M2e proteins and conferring effective mucosal and systemic immune responses in the intestine as well as in the upper respiratory airways (trachea) of chickens. In addition, lavages collected from trachea and intestine of birds administered with rL. lactis expressing influenza NA or M2e protein were found to protect MDCK cells against avian influenza type A/PR/8/34 (H1N1) virus challenge. Although minor, the differences in the expression of pro-inflammatory cytokines gene transcripts targeted in this study among the birds administered with either empty or rL. lactis could be attributed to the activation of innate response by L. lactis.

Evaluation of recombinant adenovirus vectors and adjuvanted protein as a heterologous prime-boost strategy using HER2 as a model antigen

Induction of strong antigen-specific cell-mediated and humoral responses are critical to developing a successful therapeutic vaccine. Herein, using HER2 as a model antigen, we aim to evaluate a therapeutic vaccine protocol that elicits anti-tumor antibody and cytotoxic T cells to HER2/neu antigen. Replication-competent (ΔPS AdV) and non-replicating recombinant adenoviral vectors (AdV) expressing a rat HER2/neu (ErbB2) oncogene, were generated and compared for four different doses and over four time points for their ability to induce antigen-specific T and B cell responses in mice. Although ΔPS AdV:Her2 vector was shown to induce more durable antigen-specific CD8+ T cell responses, overall, the AdV:Her2 vector induced broader T and B cell responses. Hence the AdV:Her2 vector was used to evaluate a heterologous prime-boost vaccination regimen using rat HER2 protein encapsulated in archaeosomes composed of a semi-synthetic glycolipid (sulfated S-lactosylarchaeol, SLA; and lactosylarchaeol, LA) (SLA/LA:HER2enc) or admixed with archaeosomes composed of SLA alone (SLA:HER2adm). We first tested AdV:Her2 using a prime-boost approach with SLA/LA:HER2enc, and thereafter evaluated a sub-optimal AdV:Her2 dose in a heterologous prime-boost approach with SLA:HER2adm. A single administration of AdV:Her2 alone induced strong cell-mediated immune responses, whereas SLA/LA:HER2enc alone induced strong antigen-specific IgG titers. In mice primed with a suboptimal dose of AdV:Her2, strong CD8+ T-cell responses were observed after a single dose which were not further augmented by protein boost. AdV:Her2 induced CD4+ specific T-cell responses were augmented by SLA:HER2adm. Homologous vaccination using SLA:HER2adm induced strong antigen-specific antibody responses. However, the overall magnitude of the responses was similar with three doses of SLA:HER2adm or Ad:HER2 prime followed by two doses of SLA:HER2adm.We demonstrate that AdV:Her2 is capable of inducing strong antigen-specific CD8+ T cell responses, even at a low dose, and that these responses can be broadened to include antigen-specific antibody responses by boosting with SLA adjuvanted proteins without compromising CD8 T cell responses elicited by AdV priming.

Human TIGIT on porcine aortic endothelial cells suppresses xenogeneic macrophage-mediated cytotoxicity

PurposeThe delayed rejection caused by strong cell-mediated innate and adaptive xenogeneic immune responses continues to be a major obstacle.Therefore, suppressing macrophage function could be effective in avoiding this type of rejection. In this study, the suppression of T-cell immunoglobulin and ITIM domain (TIGIT) function against macrophage-mediated xenogeneic rejection was investigated. Material and methodsNaïve porcine aortic endothelial cell (PAEC) and PAEC transfectant with TIGIT (PAEC/TIGIT) were co-cultured with M1 macrophages, and the degree of cytotoxicity was determined by a counting beads assay. The anti/pro-inflammatory gene expression was determined by RT-PCR and the phosphorylated SHP-1 in the macrophages after co-culturing with PAEC or PAEC/TIGIT was evaluated by western blotting. ResultsCD155 was expressed at essentially equal levels on both M1 and M2 macrophages, whereas TIGIT was highly expressed on M2 macrophages but not in M1 macrophages. TIGIT on PAEC significantly reduced the cytotoxicity of M1 macrophages but no significant suppression of phagocytosis was detected. TIGIT also caused a decrease in the expression of pro-inflammatory cytokines, namely TNFα, IL-1β and IL-12 in M1 macrophages. Furthermore, PAEC/TIGIT caused a significant increase in phosphorylated SHP-1 in M1 macrophages compared to PAEC. ConclusionThe findings of this study indicate that TIGIT suppresses xenogeneic M1 macrophage-induced cytotoxicity, probably at least in part, via the phosphorylation of SHP-1. In addition, the reduced expression of some pro-inflammatory cytokines, namely TNFα, IL-1β and IL-12, was observed in M1 macrophages that had been cultured with PAEC/TIGIT.

A comparison of the immune responses induced by antigens in three different archaeosome-based vaccine formulations

Archaeosomes are liposomes composed of natural or synthetic archaeal lipids that can be used as adjuvants to induce strong long-lasting humoral and cell-mediated immune responses against entrapped antigen. However, the entrapment efficiency of antigen within archaeosomes constituted using standard liposome forming methodology is often only 5–40%. In this study, we evaluated different formulation methods using a simple semi-synthetic archaeal lipid (SLA, sulfated lactosyl archaeol) and two different antigens, ovalbumin (OVA) and hepatitis B surface antigen (HBsAg). Antigen was entrapped within archaeosomes using the conventional thin film hydration-rehydration method with or without removal of non-entrapped antigen, or pre-formed empty archaeosomes were simply admixed with an antigen solution. Physicochemical characteristics were determined (size distribution, zeta potential, vesicle morphology and lamellarity), as well as location of antigen relative to bilayer using cryogenic transmission electron microscopy (TEM). We demonstrate that antigen (OVA or HBsAg) formulated with SLA lipid adjuvants using all the different methodologies resulted in a strong antigen-specific immune response. Nevertheless, the advantage of using a drug substance process that comprises of simply admixing antigen with pre-formed empty archaeosomes, represents a simple, efficient and antigenic dose-sparing formulation for adjuvanting and delivering vaccine antigens.

Acute inflammation and influenza: Innovative nanoparticle vaccination studies in a pig model

According to the reports of World Health Organization (WHO), around three to five million people are infected with seasonal influenza annually and approximately 250,000 to 500,000 people die worldwide. The risk of influenza associated complications are due to severe inflammatory reactions in the respiratory tract, and it is common in children <5 years, pregnant women, elderly individuals and people with chronic medical conditions. Influenza A virus (IAV) subtypes H1N1 and H3N2 are the major currently circulating viruses among humans. Vaccination is the most effective way to prevent influenza in humans. Influenza virus primarily infects epithelial cells lining the respiratory tract mucosa, and nasal virus shedding forms the means of viral transmission. Hence, induction of strong mucosal antibody and cell-mediated immune responses are beneficial for efficient protection during influenza epidemics and pandemics. But protection from currently used influenza vaccines varies from 10 to 60%, and it induces poor mucosal immune responses. Since inflammation associated with IAV infection is a ‘double edge sword’, control of infection-associated morbidity is essential. It is possible only through the use of potent intranasal influenza vaccines, which induce robust mucosal immune response and alleviates inflammation in the respiratory tract. This review article summarizes inflammatory responses triggered by IAV infection causing severe pulmonary disease, and the novel mucosal vaccination strategies, tested in a pig model, that have been showing promise to mitigate influenza-induced immunopathology.

Induction of immune responses by a novel recombinant fusion protein of enterovirus A71 in BALB/c mice

Fusion protein technology is used in biotechnology and medical developments. In this study, recombinant fusion proteins from enterovirus A71 (EV-A71) subgenotype B5, Thailand were designed based two surface proteins (VP1 and VP2) and an internal protein (VP4), and named “VP0” (consisting of VP4-VP2) and “EV71” (consisting of VP4-VP2-VP1), respectively. The recombinant fusion proteins VP0 and EV71 were expressed in insect cells and successfully produced and secreted into the media. Both recombinant fusion proteins were shown to have immunogenic properties in BALB/c mice when formulated with Freund’s complete/incomplete adjuvant (FA). Interestingly, EV71 formulated with FA- induced a level of IgG antibodies level similar to that induced by the recombinant protein VP1 formulated with FA (the positive control). Our results showed that VP1 alone is better at eliciting a strong cell-mediated immune response. Nontheless, EV71 formulated with FA was capable of inducing lymphocyte proliferation and increasing the cytokine-related mRNA expression levels of interferon-γ (IFN-γ), interleukin-2 (IL-2), and IL-10 in mice after immunization. Additionally, the number of CD4+ and CD8+ T lymphocyte cells after stimulation with purified EV71 in splenic cell culture showed highly specific CD4+ and CD8+ T-cell production. We suggest that EV71, which consists of VP4-VP2-VP1, could be used as the foundation for developing a novel recombinant fusion protein-based vaccine for EV-A71.

Atlantic salmon post-smolts adapted for a longer time to seawater develop an effective humoral and cellular immune response against Salmonid alphavirus

Salmonid alphavirus (SAV) causes pancreas disease (PD) in Atlantic salmon (Salmo salar L.) and disease outbreaks are mainly detected after seawater transfer. The influence of the smoltification process on the immune responses, specifically the adaptive response of Atlantic salmon after SAV infection, is not fully understood. In this study, Atlantic salmon post-smolts were infected by either bath immersion (BI) or intramuscular injection (IM) with SAV subtype 3, 2 weeks (Phase A) or 9 weeks (Phase B) after seawater transfer. The transcript levels of genes related to cellular, humoral and inflammatory responses were evaluated on head kidney samples collected at 3, 7, 14, 21, and 28 days post-infection (dpi). Corresponding negative control groups (CT) were established accordingly. Significant differences were found between both phases and between the IM and BI groups. The anti-inflammatory cytokine IL-10 was up-regulated in Phase A at a higher level than in Phase B. High mRNA levels of the genes RIG-1, SOCS1 and STAT1 were observed in all groups except the BI-B group (BI-Phase B). Moreover, the IM-B group showed a higher regulation of genes related to cellular responses, such as CD40, MHCII, and IL-15, that indicated the activation of a strong cell-mediated immune response. CD40 mRNA levels were elevated one week earlier in the BI-B group than in the BI-A group (BI-Phase A). A significant up-regulation of IgM and IgT genes was seen in both IM groups, but the presence of neutralizing antibodies to SAV was detected only in Phase B fish at 21 and 28 dpi. In addition, we found differences in the basal levels of some of the analysed genes between non-infected control groups of both phases. Findings suggest that Atlantic salmon post-smolts adapted for a longer time to seawater before they come into contact with SAV, developed a stronger humoral and cell-mediated immune response during a SAV infection.

Efficient co-delivery of neo-epitopes using dispersion-stable layered double hydroxide nanoparticles for enhanced melanoma immunotherapy

Cancer immunotherapy has shown tremendous progresses in recent years for various cancers and layered double hydroxide (LDH) nanoparticles are demonstrated as effective adjuvants for protein-based vaccines. This research further shows that the colloidal stability of LDH-based vaccines significantly influences the therapeutic efficacy and LDH nanoparticles are able to adjuvant multiple tumor-associated antigen peptides to provoke strong cell-mediated immune responses for effective inhibition of cancer growth. The LDH-based multi-target therapeutic vaccines were constructed by assembling epitope peptides and CpG onto LDH nanoparticles. Using melanoma as the model cancer and Tyrosinase-related protein 2 (Trp2) peptide as the model antigen, we demonstrated that dispersion-stable LDH-based vaccine induced stronger cytotoxic T-lymphocyte (CTL) responses and significantly inhibited tumor growth in comparison with aggregated LDH-based vaccine. We further constructed multi-target dispersion-stable LDH-based vaccine by co-loading Trp2, two mutated epitopes (M27 and M30) and CpG, which showed remarkable inhibition of melanoma growth. These results suggest that dispersion-stable LDH nanoparticles are an ideal platform to load multi-antigens and immune stimulants as effective personalized therapeutic cancer vaccines.

Immunoinformatics approaches to design a novel multi-epitope subunit vaccine against HIV infection

The end goal of HIV vaccine designing requires novel strategies to elicit a strong humoral and cell-mediated immune response. The emergence of drug resistance and the requirement of next line treatment necessitate the finding of the potential and immunogenic vaccine candidate. This study employed a novel immunoinformatics approach to design multi-epitope subunit vaccine against HIV infection. Here, we designed the subunit vaccine by the combination of CTL, HTL and BCL epitopes along with suitable adjuvant and linkers. Physiochemical characterization of subunit vaccine was assessed to ensure its thermostability, theoretical PI, and amphipathic behavior. In further assessment, subunit vaccine was found to be immunogenic with the capability to generate humoral and cell-mediated immune response. Further, homology modeling and refinement was performed and the refined modeled structure was used for molecular docking with the immune receptor (TLR-3) present on lymphocyte cells. Consequently, molecular dynamics simulation ensured the molecular interaction between TLR-3 and subunit vaccine candidate. Disulfide engineering was performed by placing the cysteine residues in the region of high mobility to enhance the vaccine stability. At last, in silico cloning was performed to warrant the translational efficiency and microbial expression of the designed vaccine.

Immune response profiles of calves following vaccination with live BCG and inactivated Mycobacterium bovis vaccine candidates.

Conventional control and eradication strategies for bovine tuberculosis (BTB) face tremendous difficulties in developing countries; countries with wildlife reservoirs, a complex wildlife-livestock-human interface or a lack of veterinary and veterinary public health surveillance. Vaccination of cattle and other species might in some cases provide the only suitable control strategy for BTB, while in others it may supplement existing test-and-slaughter schemes. However, the use of live BCG has several limitations and the global rise of HIV/AIDS infections has furthermore warranted the exploration of inactivated vaccine preparations. The aim of this study was to compare the immune response profiles in response to parenteral vaccination with live BCG and two inactivated vaccine candidates in cattle.Twenty-four mixed breed calves (Bos taurus) aged 4–6 months, were allocated to one of four groups and vaccinated sub-cutaneously with live M. bovis BCG (Danish 1331), formalin-inactivated M. bovis BCG, heat-killed M. bovis or PBS/Montanide™ (control). Interferon-γ responsiveness and antibody production were measured prior to vaccination and at weekly intervals thereafter for twelve weeks. At nine weeks post-priming, animals were skin tested using tuberculins and MTBC specific protein cocktails and subsequently challenged through intranodular injection of live M. bovis BCG.The animals in the heat-killed M. bovis group demonstrated strong and sustained cell-mediated and humoral immune responses, significantly higher than the control group in response to vaccination, which may indicate a protective immune profile. Animals in this group showed reactivity to the skin test reagents, confirming good vaccine take. Lastly, although not statistically significant, recovery of BCG after challenge was lowest in the heat-killed M. bovis group.In conclusion, the parenteral heat-killed M. bovis vaccine proved to be clearly immunogenic in cattle in the present study, urging further evaluation of the vaccine in challenge studies using virulent M. bovis and assessment of vaccine efficacy in field conditions.

Expression and Immunogenicity of Two Recombinant Fusion Proteins Comprising Foot-and-Mouth Disease Virus Structural Protein VP1 and DC-SIGN-Binding Glycoproteins.

Improving vaccine immunogenicity by targeting antigens to dendritic cells has recently emerged as a new design strategy in vaccine development. In this study, the VP1 gene of foot-and-mouth disease virus (FMDV) serotype A was fused with the gene encoding human immunodeficiency virus (HIV) membrane glycoprotein gp120 or C2-V3 domain of hepatitis C virus (HCV) envelope glycoprotein E2, both of which are DC-SIGN-binding glycoproteins. After codon optimization, the VP1 protein and the two recombinant VP1-gp120 and VP1-E2 fusion proteins were expressed in Sf9 insect cells using the insect cell-baculovirus expression system. Western blotting showed that the VP1 protein and two recombinant VP1-gp120 and VP1-E2 fusion proteins were correctly expressed in the Sf9 insect cells and had good reactogenicity. Guinea pigs were then immunized with the purified proteins, and the resulting humoral and cellular immune responses were analyzed. The VP1-gp120 and VP1-E2 fusion proteins induced significantly higher specific anti-FMDV antibody levels than the VP1 protein and stronger cell-mediated immune responses. This study provides a new perspective for the development of novel FMDV subunit vaccines.

Superior Protection from Live-Attenuated Vaccines Directed against Johne's Disease.

Mycobacterium avium subsp. paratuberculosis (M. paratuberculosis) is the etiological agent of Johne's disease in ruminants. Johne's disease is an important enteric infection causing large economic losses associated with infected herds. In an attempt to fight this infection, we created two novel live-attenuated vaccine candidates with mutations in sigH and lipN (pgsH and pgsN, respectively). Earlier reports in mice suggested these vaccines are promising candidates to fight Johne's disease in ruminants. In this study, we tested the performances of the two constructs as vaccine candidates using the goat model of Johne's disease. Both vaccines appeared to provide significant immunity to goats against challenge from wild-type M. paratuberculosis The pgsH and pgsN constructs showed a significant reduction in histopathological lesions and tissue colonization compared to nonvaccinated goats and those vaccinated with an inactivated vaccine. Unlike the inactivated vaccine, the pgsN construct was able to eliminate fecal shedding from challenged animals, a feature that is highly desirable to control Johne's disease in infected herds. Furthermore, strong initial cell-mediated immune responses were elicited in goats vaccinated with pgsN that were not demonstrated in other vaccine groups. Overall, the results indicate the potential use of live-attenuated vaccines to control intracellular pathogens, including M. paratuberculosis, and warrant further testing in cattle, the main target for Johne's disease control programs.

Virulence Associated Genes-Deleted Salmonella Montevideo Is Attenuated, Highly Immunogenic and Confers Protection against Virulent Challenge in Chickens.

To construct a novel live vaccine against Salmonella enterica serovar Montevideo (SM) infection in chickens, two important bacterial regulatory genes, lon and cpxR, which are associated with invasion and virulence, were deleted from the wild type SM genome. Attenuated strains, JOL1625 (Δlon), JOL1597 (ΔcpxR), and JOL1599 (ΔlonΔcpxR) were thereby generated. Observations with scanning electron microscopy suggested that JOL1625 and JOL1599 cells showed increased ruffled surface which may be related to abundant extracellular polysaccharide (EPS) production. JOL1597 depicted milder ruffled surface but showed increased surface corrugation. ConA affinity-based fluorometric quantification and fluorescence microscopy revealed significant increases in EPS production in JOL1625 and JOL1599. Four weeks old chickens were used for safety and immunological studies. The mutants were not observed in feces beyond day 3 nor in spleen and cecum beyond day 7, whereas wild type SM was detected for at least 2 weeks in spleen and cecum. JOL1599 was further evaluated as a vaccine candidate. Chickens immunized with JOL1599 showed strong humoral responses, as indicated by systemic IgG and secretory IgA levels, as well as strong cell-mediated immune response, as indicated by increased lymphocyte proliferation. JOL1599-immunized groups also showed significant degree of protection against wild type challenge. Our results indicate that Δlon- and/or ΔcpxR-deleted SM exhibited EPS-enhanced immunogenicity and attenuation via reduced bacterial cell intracellular replication, conferred increased protection, and possess safety qualities favorable for effective vaccine development against virulent SM infections.

Combination of Adt-O1Manisa and Ad5-boIFNλ3 induces early protective immunity against foot-and-mouth disease in cattle

Foot-and-mouth-disease (FMD) remains the most infectious livestock disease worldwide. Although commercially available inactivated or adenovirus-vectored-vaccines (Ad5-FMD) are effective, they require 5–7 days to induce protection. Therefore, new control strategies that stimulate rapid immune responses are needed. Expression of bovine interferon λ3 using the Ad5-vector platform (Ad5-boIFNλ3) is able to delay disease in cattle, but clinical signs appear at 9 days after challenge. We hypothesized that combination of Ad5-boIFNλ3 and Ad5-FMD could induce immediate and lasting protection against FMD. Cattle were vaccinated with an Ad5-FMD, Ad5-boIFNλ3, or the combination of both, followed by challenge at three days post-immunization. All animals treated with Ad5-FMD combined with Ad5-boIFNλ3 were fully protected against FMD, despite the absence of systemic neutralizing antibodies or antiviral activity at the time of challenge. Induction of a strong cell-mediated immune response suggested that Ad5-boIFNλ3 is able to act as an adjuvant of Ad5-FMD vaccine in cattle.

Human immune response to salivary proteins of wild-caught Phlebotomus papatasi.

Phlebotomine sand flies are the known vectors of Leishmania parasites. New approaches in vaccination against Leishmania have investigated the possibility of integrating Phlebotomus papatasi salivary proteins to enhance the immune response and protect against the transmission of the infection. The aim of the present study was to screen human immune responses to wild sand fly saliva and evaluate immunogenic salivary proteins. Blood samples were collected from donors in control and sand fly infested areas. Antibodies specific for sand fly antigens in donor plasma were probed using immunoblotting. In addition, recall proliferation capability of peripheral blood mononuclear cells (PBMC) was tested after sand fly salivary homogenates stimulation. The significant immunogenic salivary proteins (SPs) identified by immunoblotting were SP28, SP32, and SP36. A specific proliferative response of PBMC after stimulation with sand fly salivary homogenates was evident in donors that have antibody responses against sand fly salivary proteins. Individuals with antibody recognition to a higher number of salivary proteins (i.e., 3 or more SP bands) showed lower PBMC proliferative responses after in vitro stimulation with salivary gland homogenates (SGH) only in the sand fly infested, leishmaniasis free area. Interestingly, the presence of a humoral immune response to many SP antigens inversely correlates with a strong cell-mediated immune response (CMI). It was also noticed that some other heavily expressed antigens, in sand fly salivary homogenate, lack or have weak humoral immune reactivity in exposed individuals. Therefore, considering these antigens alone as CMI activators, without including the immunodominant humoral immune response proteins, needs future investigation.