Potent Protective Immune Responses Research Articles

Probiotic-Based Vaccines May Provide Effective Protection against COVID-19 Acute Respiratory Disease.

Severe acute respiratory syndrome coronavirus 2 virus (SARS-CoV-2) infection, the causative agent of COVID-19, now represents the sixth Public Health Emergency of International Concern (PHEIC)—as declared by the World Health Organization (WHO) since 2009. Considering that SARS-CoV-2 is mainly transmitted via the mucosal route, a therapy administered by this same route may represent a desirable approach to fight SARS-CoV-2 infection. It is now widely accepted that genetically modified microorganisms, including probiotics, represent attractive vehicles for oral or nasal mucosal delivery of therapeutic molecules. Previous studies have shown that the mucosal administration of therapeutic molecules is able to induce an immune response mediated by specific serum IgG and mucosal IgA antibodies along with mucosal cell-mediated immune responses, which effectively concur to neutralize and eradicate infections. Therefore, advances in the modulation of mucosal immune responses, and in particular the use of probiotics as live delivery vectors, may encourage prospective studies to assess the effectiveness of genetically modified probiotics for SARS-CoV-2 infection. Emerging trends in the ever-progressing field of vaccine development re-emphasize the contribution of adjuvants, along with optimization of codon usage (when designing a synthetic gene), expression level, and inoculation dose to elicit specific and potent protective immune responses. In this review, we will highlight the existing pre-clinical and clinical information on the use of genetically modified microorganisms in control strategies against respiratory and non-respiratory viruses. In addition, we will discuss some controversial aspects of the use of genetically modified probiotics in modulating the cross-talk between mucosal delivery of therapeutics and immune system modulation.

A proof of concept for neutralizing antibody-guided vaccine design against SARS-CoV-2.

Mutations and transient conformational movements of the receptor binding domain (RBD) that make neutralizing epitopes momentarily unavailable present immune escape routes for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). To mitigate viral escape, we developed a cocktail of neutralizing antibodies (NAbs) targeting epitopes located on different domains of spike (S) protein. Screening of a library of monoclonal antibodies generated from peripheral blood mononuclear cells of COVID-19 convalescent patients yielded potent NAbs, targeting the N-terminal domain (NTD) and RBD domain of S, effective at nM concentrations. Remarkably, a combination of RBD-targeting NAbs and NTD-binding NAbs, FC05, enhanced the neutralization potency in cell-based assays and an animal model. Results of competitive surface plasmon resonance assays and cryo-electron microscopy (cryo-EM) structures of antigen-binding fragments bound to S unveil determinants of immunogenicity. Combinations of immunogens, identified in the NTD and RBD of S, when immunized in rabbits and macaques, elicited potent protective immune responses against SARS-CoV-2. More importantly, two immunizations of this combination of NTD and RBD immunogens provided complete protection in macaques against a SARS-CoV-2 challenge, without observable antibody-dependent enhancement of infection. These results provide a proof of concept for neutralization-based immunogen design targeting SARS-CoV-2 NTD and RBD.

Immunogenicity study of OmpU subunit vaccine against Vibrio mimicus in yellow catfish, Pelteobagrus fulvidraco

The outer membrane protein U (OmpU) is a conserved outer membrane protein in a variety of pathogenic Vibrio species and has been considered as a vital protective antigen for vaccine development. Vibrio mimicus (V. mimicus) is the pathogen causing ascites disease in aquatic animals. In this study, the prokaryotically expressed and purified His-tagged OmpU of V. mimicus (His-OmpU) was used as a subunit vaccine. The formalin inactivated V. mimicus, purified His tag (His-tag), and PBS were used as controls. The vaccinated yellow catfish were challenged with V. mimicus at 28 days post-vaccination, and the results showed that the His-OmpU and inactivated V. mimicus groups exhibited much higher survival rates than the His-tag and PBS groups. To fully understand the underlying mechanism, we detected the expression levels of several immune-related genes in the spleen of fish at 28 days post-vaccination and 24 h post-challenge. The results showed that most of the detected immune-related genes were significantly upregulated in His-OmpU and inactivated V. mimicus groups. In addition, we performed the serum bactericidal activity assay, and the results showed that the serum from His-OmpU and inactivated V. mimicus groups exhibited much stronger bactericidal activity against V. mimicus than those of His-tag and PBS groups. Finally, the serum agglutination antibody was detected, and the antibody could be detected in His-OmpU and inactivated V. mimicus groups with the antibody titers increasing along with the time post-vaccination, but not in His-tag or PBS group. Our data reveal that the recombinant OmpU elicits potent protective immune response and is an effective vaccine candidate against V. mimicus in yellow catfish.

Potent Protective Immune Responses to Senecavirus Induced by Virus-Like Particle Vaccine in Pigs.

Senecavirus A (SVA) is the pathogen that has recently caused porcine idiopathic vesicular disease (PIVD). The clinical symptoms of PIVD are similar to those of acute foot-and-mouth disease and also can result in the death of newborn piglets, thus entailing economic losses. Vaccine immunization is the most effective way to prevent and control SVA. Among all SVA vaccines reported, only the SVA inactivated vaccine has been successfully developed. However, to ensure the elimination of this pathogen, safer and more effective vaccines are urgently required. A virus-like particles (VLPs)-based vaccine is probably the best alternative to inactivated vaccine. To develop an SVA VLPs vaccine and evaluate its immune effect, a prokaryotic expression system was used to produce SVA capsid protein and assemble VLPs. The VLPs were characterized by affinity chromatography, sucrose density gradient centrifugation, ZetaSizer and transmission electron microscopy. Meanwhile, the SVA CH-HB-2017 strain was used to infect pigs and to determine infection routes and dose. Experimental pigs were then immunized with the SVA VLPs vaccine emulsified in an ISA 201 adjuvant. The results showed that the VLPs vaccine induced neutralizing and specific antibodies at similar levels as an inactivated SVA vaccine after immunization. The level of INF-γ induced by the VLPs vaccine gradually decreased—similar to that of inactivated vaccine. These results indicated that VLPs vaccine may simultaneously cause both cellular and humoral immune responses. Importantly, after the challenge, the VLPs vaccine provided similar levels of protection as the inactivated SVA vaccine. In this study, we successfully obtained novel SVA VLPs and confirmed their highly immunogenicity, thus providing a superior candidate vaccine for defense and elimination of SVA, compared to the inactivated vaccine.

Innovative Mucosal Vaccine Formulations Against Influenza A Virus Infections.

Despite efforts made to develop efficient preventive strategies, infections with influenza A viruses (IAV) continue to cause serious clinical and economic problems. Current licensed human vaccines are mainly inactivated whole virus particles or split-virion administered via the parenteral route. These vaccines provide incomplete protection against IAV in high-risk groups and are poorly/not effective against the constant antigenic drift/shift occurring in circulating strains. Advances in mucosal vaccinology and in the understanding of the protective anti-influenza immune mechanisms suggest that intranasal immunization is a promising strategy to fight against IAV. To date, human mucosal anti-influenza vaccines consist of live attenuated strains administered intranasally, which elicit higher local humoral and cellular immune responses than conventional parenteral vaccines. However, because of inconsistent protective efficacy and safety concerns regarding the use of live viral strains, new vaccine candidates are urgently needed. To prime and induce potent and long-lived protective immune responses, mucosal vaccine formulations need to ensure the immunoavailability and the immunostimulating capacity of the vaccine antigen(s) at the mucosal surfaces, while being minimally reactogenic/toxic. The purpose of this review is to compile innovative delivery/adjuvant systems tested for intranasal administration of inactivated influenza vaccines, including micro/nanosized particulate carriers such as lipid-based particles, virus-like particles and polymers associated or not with immunopotentiatory molecules including microorganism-derived toxins, Toll-like receptor ligands and cytokines. The capacity of these vaccines to trigger specific mucosal and systemic humoral and cellular responses against IAV and their (cross)-protective potential are considered.

Toll-like receptor 4 signalling regulates antibody response to adenoviral vector-based vaccines by imprinting germinal centre quality.

Adenoviral vectors (AdV) are considered promising candidates for vaccine applications. A prominent group of Toll-like receptors (TLRs) participate in the adenovirus-induced adaptive immune response, yet there is little information regarding the role of TLR4 in AdV-induced immune responses in recent literature. We investigated the function of TLR4 in both adaptive and innate immune responses to an AdV-based anthrax vaccine. By immunizing wild-type and TLR4 knockout (TLR4-KO) mice, we revealed the requirement of TLR4 in AdV-induced innate responses. We also showed that TLR4 functions are required for germinal centre responses in immunized mice, as expression of the apoptosis-related marker Fas was down-regulated on germinal centre B cells from TLR4-KO mice. Likewise, decreased expression of inducible costimulator on follicular T helper cells was observed in immunized TLR4-KO mice. Moreover, a potent protective antigen-specific humoral immune response was mimicked using an adjuvant system containing the TLR4 agonist monophosphoryl lipid A. Overall, our findings showed that very rapid antigen-specific antibody production is correlated with the TLR4-imprinted germinal centre response to AdV-based vaccine. These results provide additional evidence for the use of the AdV and a TLR agonist to induce humoral responses. Our findings offer new insights into rational vaccine design.

Single dose HBsAg CS-γ-PGA nanogels induce potent protective immune responses against HBV infection

Hepatitis B virus (HBV) infection is a severe threat to public health, which can be prevented by prophylactic vaccination. Here, we tested nanogels carriers in the prophylactic effect of hepatitis B surface antigen (HBsAg) vaccine. HBsAg nanogels (Ng) were prepared using chitosan (CS) and poly-γ-glutamic acid (γ-PGA). Positively charged Ng (+) and negatively charged Ng (−) were prepared by adjusting the CS and γ-PGA proportion. Dendritic cells (DCs) maturation in mice immunized with HBsAg Ng (+) and HBsAg Ng (−) could be augmented in response to pAAV/HBV1.2 plasmid challenge. Single-dose immunization with HBsAg Ng (+) induced HBsAg specific-antibodies. HBsAg Ng (+) immunized mice cleared HBsAg and restored anti-HBs production after pAAV/HBV1.2 plasmid challenge. Single-dose HBsAg Ng (+) induced humoral and cellular immunity, and could induce effector memory T cells. Single-dose HBsAg Ng (−) favored the induction of cellular immunity, and induced central memory T cells and effector memory T cells. However, HBsAg elimination was similar between HBsAg Ng (+)- and HBsAg Ng (+) plus HBsAg Ng (−)-immunized mice. Zeta potential measurements showed that HBsAg Ng (+) were more stable than HBsAg Ng (−). Therefore, Ng (+) are desirable HBsAg prophylactic vaccine carriers, providing long-term protection against HBV, and are a good choice to study and apply weakly immunostimulatory antigens.

Liposomes containing monophosphoryl lipid A and QS-21 serve as an effective adjuvant for soluble circumsporozoite protein malaria vaccine FMP013

Malaria caused by Plasmodium falciparum continues to threaten millions of people living in the tropical parts of the world. A vaccine that confers sterile and life-long protection remains elusive despite more than 30years of effort and resources invested in solving this problem. Antibodies to a malaria vaccine candidate circumsporozoite protein (CSP) can block invasion and can protect humans against malaria. We have manufactured the Falciparum Malaria Protein-013 (FMP013) vaccine based on the nearly full-length P. falciparum CSP 3D7 strain sequence. We report here immunogenicity and challenge data on FMP013 antigen in C57BL/6 mice formulated with two novel adjuvants of the Army Liposome Formulation (ALF) series and a commercially available adjuvant Montanide ISA 720 (Montanide) as a control. ALF is a liposomal adjuvant containing a synthetic monophosphoryl lipid A (3D-PHAD®). In our study, FMP013 was adjuvanted with ALF alone, ALF containing aluminum hydroxide (ALFA) or ALF containing QS-21 (ALFQ). Adjuvants ALF and ALFA induced similar antibody titers and protection against transgenic parasite challenge that were comparable to Montanide. ALFQ was superior to the other three adjuvants as it induced higher antibody titers with improved boosting after the third immunization, higher serum IgG2c titers, and enhanced protection. FMP013+ALFQ also augmented the numbers of splenic germinal center-derived activated B-cells and antibody secreting cells compared to Montanide. Further, FMP013+ALFQ induced antigen-specific IFN-γ ELISPOT activity, CD4+ T-cells and a TH1-biased cytokine profile. These results demonstrate that soluble CSP can induce a potent and sterile protective immune response when formulated with the QS-21 containing adjuvant ALFQ. Comparative mouse immunogenicity data presented here were used as the progression criteria for an ongoing non-human primate study and a regulatory toxicology study in preparation for a controlled human malaria infection (CHMI) trial.

Innate and adaptive immune responses of catfish antigen-presenting cells to live attenuated Edwardsiella ictaluri vaccines

Efficacious live attenuated Edwardsiella ictaluri vaccines are expected to deliver the antigen to antigenpresenting cells (APCs) that can elicit potent protective innate and adaptive immune responses resulting in the killing of the infected targets or inducing the killing mechanisms in the infected cells. T cell-inducing vaccines must aim to deliver the antigen to APCs so that it can be presented on MHC molecules to the specific T cells. Dendritic cells (DCs) are the most powerful APCs that bridge, control and regulate innate and adaptive immune responses in vertebrates. Recently, we have identified the presence of the Langerhanlike cells containing Birbeck granules in the hemopoietic tissues of channel catfish by the following approaches: flow cytometry, immunohistochemistry, and transmission electron microscopy. Langerin/CD207-positive cells have been detected in catfish spleen and anterior kidney but not in peripheral blood and skin by using mAbs to human Langerin/CD207. These results lay a foundation for our present study that is to determine the vaccineinduced productive innate and adaptive immune responses through catfish APCs, DCs, macrophages, and B cells. Currently, we are evaluating the possible role of Langerin/CD207-positive cells in the protective immunity to E. ictaluri vaccines by immunohistochemistry. The function of professional phagocytes, macrophages, to produce nitric oxide in response to live attenuated vaccines are being evaluated by assessing the nitric oxide synthase gene expression levels by real-time PCR. In addition, to characterize the effects of live attenuated vaccines, we are evaluating T cell-mediated immunity that controls function of APCs and by assessing the gene expression levels of IFN in peripheral blood, anterior kidney, and spleen of catfish. Finally, the effects of B cell-mediated immunity, B cell-specific gene expression, and immunoglobulin M titers are being determined to live attenuated E. ictaluri vaccines. We suggest that current research will provide the optimal vaccination regime and important basic knowledge on the mechanisms of innate and adaptive immune responses against the most effective vaccines.

Principles of Vaccination.

While many of the currently available vaccines have been developed empirically, with limited understanding on how they activate the immune system and elicit protective immunity, the recent progress in basic sciences like immunology, microbiology, genetics, and molecular biology has fostered our understanding on the interaction of microorganisms with the human immune system. In consequence, modern vaccine development strongly builds on the precise knowledge of the biology of microbial pathogens, their interaction with the human immune system, as well as their capacity to counteract and evade innate and adaptive immune mechanisms. Strategies engaged by pathogens strongly determine how a vaccine should be formulated to evoke potent and efficient protective immune responses. The improved knowledge of immune response mechanisms has facilitated the development of new vaccines with the capacity to defend against challenging pathogens and can help to protect individuals particular at risk like immunocompromised and elderly populations. Modern vaccine development technologies include the production of highly purified antigens that provide a lower reactogenicity and higher safety profile than the traditional empirically developed vaccines. Attempts to improve vaccine antigen purity, however, may result in impaired vaccine immunogenicity. Some of such disadvantages related to highly purified and/or genetically engineered vaccines yet can be overcome by innovative technologies, such as live vector vaccines, and DNA or RNA vaccines. Moreover, recent years have witnessed the development of novel adjuvant formulations that specifically focus on the augmentation and/or control of the interplay between innate and adaptive immune systems as well as the function of antigen-presenting cells. Finally, vaccine design has become more tailored, and in turn has opened up the potential of extending its application to hitherto not accessible complex microbial pathogens plus providing new immunotherapies to tackle diseases such as cancer, Alzheimer's disease, and autoimmune disease. This chapter gives an overview of the key considerations and processes involved in vaccine development. It also describes the basic principles of normal immune respoinses and its their function in defense of infectious agents by vaccination.

Type III interferon attenuates a vesicular stomatitis virus-based vaccine vector.

Vesicular stomatitis virus (VSV) has been extensively studied as a vaccine vector and oncolytic agent. Nevertheless, safety concerns have limited its widespread use in humans. The type III lambda interferon (IFN-λ) family of cytokines shares common signaling pathways with the IFN-α/β family and thus evokes similar antiviral activities. However, IFN-λ signals through a distinct receptor complex that is expressed in a cell type-specific manner, which restricts its activity to epithelial barriers, particularly those corresponding to the respiratory and gastrointestinal tracts. In this study, we determined how IFN-λ expression from recombinant VSV would influence vector replication, spread, and immunogenicity. We demonstrate that IFN-λ expression severely attenuates VSV in cell culture. In vivo, IFN-λ limits VSV replication in the mouse lung after intranasal administration and reduces virus spread to other organs. Despite this attenuation, however, the vector retains its capacity to induce protective CD8 T cell and antibody responses after a single immunization. These findings demonstrate a novel method of viral vector attenuation that could be used in both vaccine and oncolytic virus applications. Viruses such as VSV that are used as vaccine vectors can induce protective T cell and antibody responses after a single dose. Additionally, IFN-λ is a potent antiviral agent that has certain advantages for clinical use compared to IFN-α/β, such as fewer patient side effects. Here, we demonstrate that IFN-λ attenuates VSV replication and spread following intranasal virus delivery but does not reduce the ability of VSV to induce potent protective immune responses. These findings demonstrate that the type III IFN family may have widespread applicability for improving the safety and efficacy of viral vaccine and oncolytic vectors.

Foot-and-mouth disease virus-like particles produced by a SUMO fusion protein system in Escherichia coli induce potent protective immune responses in guinea pigs, swine and cattle

Foot-and-mouth disease virus (FMDV) causes a highly contagious infection in cloven-hoofed animals. The format of FMD virus-like particles (VLP) as a non-replicating particulate vaccine candidate is a promising alternative to conventional inactivated FMDV vaccines. In this study, we explored a prokaryotic system to express and assemble the FMD VLP and validated the potential of VLP as an FMDV vaccine candidate. VLP composed entirely of FMDV (Asia1/Jiangsu/China/2005) capsid proteins (VP0, VP1 and VP3) were simultaneously produced as SUMO fusion proteins by an improved SUMO fusion protein system in E. coli. Proteolytic removal of the SUMO moiety from the fusion proteins resulted in the assembly of VLP with size and shape resembling the authentic FMDV. Immunization of guinea pigs, swine and cattle with FMD VLP by intramuscular inoculation stimulated the FMDV-specific antibody response, neutralizing antibody response, T-cell proliferation response and secretion of cytokine IFN-γ. In addition, immunization with one dose of the VLP resulted in complete protection of these animals from homologous FMDV challenge. The 50% protection dose (PD50) of FMD VLP in cattle is up to 6.34. These results suggest that FMD VLP expressed in E. coli are an effective vaccine in guinea pigs, swine and cattle and support further development of these VLP as a vaccine candidate for protection against FMDV.

Rabies virus pathogenesis in relationship to intervention with inactivated and attenuated rabies vaccines

Despite progress in vaccine development in the past century the mechanisms behind immune responses elicited by rabies biologics or via natural infection remain largely unknown. In this study, we compared protection elicited by standard, early, or delayed prophylaxis with a reduced number of vaccine doses using inactivated and live-attenuated vaccines. Two-month-old Syrian hamsters, 4-week-old ICR mice or adult rhesus macaques were inoculated with canine rabies virus variants. Thereafter, prophylaxis was initiated 6h, 1, 2, 3, 4, 5, 6 or 7 days post-exposure (p.e.). One or several doses of inactivated (HDCV), or reverse genetically attenuated (live), or gamma-irradiated (inactivated)-ERAG333 vaccines were administered intramuscularly. The dynamics of virus spread were measured over time in the rodent models. Rabies virus reached the spinal cord at day 4 and brain at day 6 p.e. All hamsters succumbed in groups in which live ERAG333 was delayed until days 5 and 6 p.e. However, 78%, 44%, 56% and 22% of hamsters survived when one dose of live ERAG333 was administered 6h, 1, 2, 3, and 4 days p.e., respectively. Similarly, 67% survived when inactivated ERAG333 was administered at 24h p.e. All hamsters succumbed when standard prophylaxis (the Essen regimen) was delayed until days 3–6, but 67% and 33% of hamsters survived when PEP began 1 or 2 days p.e., respectively. Macaques were protected by one dose of attenuated ERAG333 at 24h p.e. The highly attenuated (live) and inactivated ERAG333 vaccines elicited potent protective immune responses, even when prophylaxis initiation was delayed. When 2–5 doses of commercial vaccine and HRIG were administered according to the Essen scheme, 89–100% of the animals survived. Reduced vaccine schedules provided efficacious intervention, regardless of the total number of vaccine doses administered.

Dietary supplementation of mannan-oligosaccharide enhances neonatal immune responses in chickens during natural exposure to Eimeria spp

BackgroundControl and eradication of intestinal infections caused by protozoa are important biomedical challenges worldwide. Prophylactic control of coccidiosis has been achieved with the use of anticoccidial drugs; however, the increase in anticoccidial resistance has raised concerns about the need for new alternatives for the control of coccidial infections. In fact, new strategies are needed to induce potent protective immune responses in neonatal individuals.MethodsThe effects of a dietary supplementation of mannan-oligosaccharide (yeast cell wall; YCW) on the local, humoral and cell-mediated immune responses, and intestinal replication of coccidia were evaluated in a neonatal animal model during natural exposure to Eimeria spp. A total of 840 one-day-old chicks were distributed among four dietary regimens: A) Control diet (no YCW) plus anticoccidial vaccine); B) Control diet plus coccidiostat; C) YCW diet plus anticoccidial vaccination; and D) YCW diet plus coccidiostat. Weight gain, feed consumption and immunological parameters were examined within the first seven weeks of life.ResultsDietary supplementation of 0.05% of YCW increased local mucosal IgA secretions, humoral and cell-mediated immune responses, and reduced parasite excretion in feces.ConclusionDietary supplementation of yeast cell wall in neonatal animals can enhance the immune response against coccidial infections. The present study reveals the potential of YCW as adjuvant for modulating mucosal immune responses.

Killed but Metabolically Active Bacillus anthracis Vaccines Induce Broad and Protective Immunity against Anthrax

Bacillus anthracis is the causative agent of anthrax. We have developed a novel whole-bacterial-cell anthrax vaccine utilizing B. anthracis that is killed but metabolically active (KBMA). Vaccine strains that are asporogenic and nucleotide excision repair deficient were engineered by deleting the spoIIE and uvrAB genes, rendering B. anthracis extremely sensitive to photochemical inactivation with S-59 psoralen and UV light. We also introduced point mutations into the lef and cya genes, which allowed inactive but immunogenic toxins to be produced. Photochemically inactivated vaccine strains maintained a high degree of metabolic activity and secreted protective antigen (PA), lethal factor, and edema factor. KBMA B. anthracis vaccines were avirulent in mice and induced less injection site inflammation than recombinant PA adsorbed to aluminum hydroxide gel. KBMA B. anthracis-vaccinated animals produced antibodies against numerous anthrax antigens, including high levels of anti-PA and toxin-neutralizing antibodies. Vaccination with KBMA B. anthracis fully protected mice against challenge with lethal doses of toxinogenic unencapsulated Sterne 7702 spores and rabbits against challenge with lethal pneumonic doses of fully virulent Ames strain spores. Guinea pigs vaccinated with KBMA B. anthracis were partially protected against lethal Ames spore challenge, which was comparable to vaccination with the licensed vaccine anthrax vaccine adsorbed. These data demonstrate that KBMA anthrax vaccines are well tolerated and elicit potent protective immune responses. The use of KBMA vaccines may be broadly applicable to bacterial pathogens, especially those for which the correlates of protective immunity are unknown.

Mechanisms of Immunization Against Cancer Using Chimeric Antigens

Successful approaches to tumor immunotherapy must overcome the physiological state of tolerance of the immune system to self-tumor antigens. Immunization with appropriate variants of syngeneic antigens can achieve this. However, improvements in vaccine design are needed for efficient cancer immunotherapy. Here we explore nine different chimeric vaccine designs, in which the antigen of interest is expressed as an in-frame fusion with polypeptides that impact antigen processing or presentation. In DNA immunization experiments in mice, three of nine fusions elevated relevant CD8(+) T-cell responses and tumor protection relative to an unfused melanoma antigen. These fusions were: Escherichia coli outer membrane protein A (OmpA), Pseudomonas aeruginosa exotoxin A, and VP22 protein of herpes simplex virus-1. The gains of immunogenicity conferred by the latter two are independent of epitope presentation by major histocompatibility complex class II (MHC II). This finding has positive implications for immunotherapy in individuals with CD4(+) T-cell deficiencies. We present evidence that antigen instability is not a sine qua non condition for immunogenicity. Experiments using two additional melanoma antigens identified different optimal fusion partners, thereby indicating that the benefits of fusion vectors remain antigen specific. Therefore large fusion vector panels such as those presented here can provide information to promote the successful advancement of gene-based vaccines.

MHC Class II-Associated Invariant Chain Linkage of Antigen Dramatically Improves Cell-Mediated Immunity Induced by Adenovirus Vaccines

The ideal vaccine induces a potent protective immune response, which should be rapidly induced, long-standing, and of broad specificity. Recombinant adenoviral vectors induce potent Ab and CD8+ T cell responses against transgenic Ags within weeks of administration, and they are among the most potent and versatile Ag delivery vehicles available. However, the impact of chronic infections like HIV and hepatitis C virus underscore the need for further improvements. In this study, we show that the protective immune response to an adenovirus-encoded vaccine Ag can be accelerated, enhanced, broadened, and prolonged by tethering of the rAg to the MHC class II-associated invariant chain (Ii). Thus, adenovirus-vectored vaccines expressing lymphocytic choriomeningitis virus (LCMV)-derived glycoprotein linked to Ii increased the CD4+ and CD8+ T cell stimulatory capacity in vitro and in vivo. Furthermore, mice vaccinated with a single dose of adenovirus-expressing LCMV-derived glycoprotein linked to Ii were protected against lethal virus-induced choriomeningitis, lethal challenge with strains mutated in immunodominant T cell epitopes, and systemic infection with a highly invasive strain. In therapeutic tumor vaccination, the vaccine was as efficient as live LCMV. In comparison, animals vaccinated with a conventional adenovirus vaccine expressing unmodified glycoprotein were protected against systemic infection, but only temporarily against lethal choriomeningitis, and this vaccine was less efficient in tumor therapy.

Immunity and protection by adoptive transfer of dendritic cells transfected with hepatitis C NS3/4A mRNA

In this study, we tested the hypothesis that adoptive transfer of dendritic cells (DCs) transfected ex vivo with mRNA encoding hepatitis C virus (HCV) NS3/4A would initiate potent HCV-specific protective immune responses in vivo. Murine DCs were transfected with NS3/4A mRNA or eGFP mRNA using either electroporation or Transmessenger Transfection Reagent and then used for adoptive transfer. Electroporation resulted in higher transfection efficiency but lower levels of eGFP and NS3/4A expression when compared to transfection with Transmessenger. The murine NS3/4A mRNA-transfected DCs were functional in T cell activation in vitro. Adoptive transfer of NS3/4A mRNA-transfected DCs resulted in migration to regional lymph nodes, strong cellular immune responses and protection from challenge with vaccinia virus expressing NS3/NS4/NS5 in mice. Furthermore, although Transmessenger mediated transfection was less efficient than electroporation in terms of number of transfected cells, the DCs transfected with NS3/4A mRNA and Transmessenger expressed higher levels of protein and induced stronger immune responses and protection than DCs transfected with NS3/4A mRNA by electroporation. Since no study has explored the in vivo efficacy of mRNA-transfected DC-mediated vaccination against viral diseases, including hepatitis C, our study provided a novel vaccination strategy against hepatitis C as well as other pathogens.

Potent protective cellular immune responses generated by a DNA vaccine encoding HSV-2 ICP27 and the E. coli heat labile enterotoxin

A mouse model was employed to evaluate protective cellular immune responses induced by an immediate early antigen of HSV-2. Particle-mediated DNA vaccination of mice with a DNA plasmid-encoding ICP27 resulted in the induction of ICP27-specific IFN-γ and TNF-α production in Balb/c mice, but little protection to intranasal challenge with wild type HSV-2. However, when the DNA vaccine was supplemented with as little as 50ng of a vector encoding the A and B subunits of the Escherichia coli heat labile enterotoxin (LT), animals were profoundly protected from morbidity and mortality. The ICP27+LT-mediated protection was correlated with a large increase in ICP27-specific IFN-γ and TNF-α production but cytokine-specific monoclonal antibody treatment at the time of challenge showed that protection was mediated predominantly by IFN-γ. Furthermore, depletion of T cell subsets prior to infectious challenge demonstrated that removal of either CD8+ or CD4+ T cells impaired protection with CD8+ T cells appearing to play a direct effector role. These data demonstrate that augmented cellular immune responses resulting from LT vector plus antigen vector administration to the skin are biologically significant, leading to enhanced protection against mucosal pathogenic challenge.

The influence of immunizing dose size and schedule on immunity to subsequent challenge with antigenically distinct strains of Eimeria maxima

Eimeria maxima, the most immunogenic of the Eimeriidae that infect the chicken, is characterized by the presence of antigenic diversity within field isolates. In priming/challenge experiments immunity to homologous infection is essentially complete while immunity against challenge by a heterologous strain is often only partial. The phenotype "escape from immune protection" is known to be influenced by both host and parasite genotypes but the impact of varied immunization dose and schedule remains poorly documented. In this manuscript we report that an immunizing dose between <or=5 and <or=20 sporulated E. maxima oocysts is consistently capable of stimulating complete (>99.99%) protective immunity against challenge by 100 oocysts of a homologous strain. In contrast, complete immunity against a heterologous strain was never observed, although increasing the immunizing dose size did frequently reduce oocyst production arising from subsequent heterologous challenge. Differences in cross-protective immunizing capacity between two strains of E. maxima were evident as the H strain consistently stimulated a more potent protective immune response than the W strain. Similarly, increasing the number of immunizing doses of the E. maxima W strain (but not the H strain) increased immune protection against subsequent heterologous challenge. When combined with previously published data the results described here suggest that the E. maxima genome encodes a pool of antigens that are capable of stimulating an immune response cross-protective against more than one strain. These antigens supplement a separate restricted pool of antigens that are capable of stimulating stronger, but strain-specific, protective immune responses.