Fusion Antigen Research Articles

Semisynthetic, self-adjuvanting vaccine development: Efficient, site-specific sortase A-mediated conjugation of Toll-like receptor 2 ligand FSL-1 to recombinant protein antigens under native conditions and application to a model group A streptococcal vaccine

Protein antigens are, in general, weakly immunogenic, and therefore require co-delivery with adjuvants to stimulate potent immune responses. The fusion of (poly)peptide antigens to immunostimulatory adjuvants (e.g. Toll-like receptor (TLR) agonists) has been demonstrated to greatly improve vaccine potency compared to mixtures of antigen and adjuvant. Chemical approaches, to enable the rapid, site-specific and high-yielding linkage of TLR2 ligands to recombinant protein antigens, have been previously optimized. These approaches require the use of denaturing conditions to ensure high reaction yields, which limits their application, as maintenance of native protein folding is necessary to elicit antibodies against conformational epitopes. Here, this work aimed to optimize an alternative method, to ensure the efficient bioconjugation of TLR2 ligands onto folded protein antigens. An enzyme-mediated approach, using Staphylococcus aureus sortase A (or a penta mutant with enhanced efficiency), was optimized for reaction yield and time, as well as enzyme type and amount. This approach enabled the site-specific conjugation of the TLR2-agonist fibroblast-stimulating lipopeptide-1 (FSL-1) onto a model group A Streptococcus (GAS) recombinant polytope antigen under conditions that maintain protein folding, yielding a homogeneous, molecularly-defined product, with ligation yields as high as 90%. Following intramuscular (IM) administration of the ligation product to humanized plasminogen AlbPLG1 mice, high-titer, antigen-specific IgG antibodies were observed, which conferred protection against subcutaneous challenge with GAS strain 5448. In comparison, mixtures of the GAS antigen with aluminum hydroxide or FSL-1 failed to provide protection, with the FSL-1 mixture yielding ~1000-fold lower antigen-specific IgG antibody titers, and the mixture with alum yielding a Th2-biased response compared to the more balanced Th1/Th2 responses observed with the FSL-1 conjugate. Overall, a FSL-1 bioconjugation method for the efficient production of antigen-TLR2 agonist conjugates, which maintain protein folding, was produced, with broad utility for the development of self-adjuvanting vaccines against subunit protein antigens.

First-In-Human Trials of GamTBvac, a Recombinant Subunit Tuberculosis Vaccine Candidate: Safety and Immunogenicity Assessment.

Tuberculosis is known to be the biggest global health problem, causing the most deaths by a single infectious agent. Vaccine-development efforts are extremely important. This paper represents the results of the first-in-human trial of recombinant subunit tuberculosis vaccine GamTBvac in a Phase I study. GamTBvac is a new BCG booster candidate vaccine containing dextran-binding domain modified Ag85a and ESAT6-CFP10 MTB antigens and CpG ODN adjuvant, formulated with dextrans. Safety and immunogenicity of GamTBvac were estimated in an open-label clinical trial on 60 Mycobacterium tuberculosis uninfected (MTB-uninfected) volunteers previously-vaccinated with Bacillus Calmette—Guérin vaccine (BCG). The candidate vaccine had an acceptable safety profile and was well-tolerated. Three different vaccine doses with a double-immunization scheme were assessed for immunogenicity and induced a significant increase in IFN-γ in-house IGRA response and IgG ELISA analysis. Among them, the half dose vaccine group (containing DBD-ESAT6-CFP10, 12.5 μg; DBD-Ag85a, 12.5 μg; CpG (ODN 2216), 75 μg; DEAE-Dextran 500 kDa, 250 μg; and Dextran 500 kDa, 5 mg) provided high, early and stable in time immune response specific to both protein antigen fusions and is proposed for the further studies.

Integrating SpyCatcher/SpyTag covalent fusion technology into phage display workflows for rapid antibody discovery

An early bottleneck in the rapid isolation of new antibody fragment binders using in vitro library approaches is the inertia encountered in acquiring and preparing soluble antigen fragments. In this report, we describe a simple, yet powerful strategy that exploits the properties of the SpyCatcher/SpyTag (SpyC/SpyT) covalent interaction to improve substantially the speed and efficiency in obtaining functional antibody clones of interest. We demonstrate that SpyC has broad utility as a protein-fusion tag partner in a eukaryotic expression/secretion context, retaining its functionality and permitting the direct, selective capture and immobilization of soluble antigen fusions using solid phase media coated with a synthetic modified SpyT peptide reagent. In addition, we show that the expressed SpyC-antigen format is highly compatible with downstream antibody phage display selection and screening procedures, requiring minimal post-expression handling with no sample modifications. To illustrate the potential of the approach, we have isolated several fully human germline scFvs that selectively recognize therapeutically relevant native cell surface tumor antigens in various in vitro cell-based assay contexts.

Adjuvant effect of enterotoxigenic Escherichia coli (ETEC) double-mutant heat-labile toxin (dmLT) on systemic immunogenicity induced by the CFA/I/II/IV MEFA ETEC vaccine: Dose-related enhancement of antibody responses to seven ETEC adhesins (CFA/I, CS1-CS6)

ABSTRACTDouble-mutant heat-labile toxin (dmLT, LTR192G/L211A) of enterotoxigenic Escherichia coli (ETEC) is an effective mucosal adjuvant. Recent studies have shown that dmLT also exhibits adjuvanticity for antigens administered parenterally. In this study, we subcutaneously (SC) immunized mice with the ETEC adhesin-based vaccine, CFA/I/II/IV MEFA (multiepitope fusion antigen), adjuvanted with dmLT and examined the impact of dmLT on antibody responses specific to the seven adhesins in the vaccine construction [CFA/I, CFA/II (CS1, CS2, CS3) and CFA/IV (CS4, CS5, CS6)]. Mice were immunized with a fixed dose of CFA/I/II/IV MEFA and ascending doses of dmLT adjuvant (0, 0.05, 0.1, 0.5 or 1.0 µg) to assess the potential dmLT dose response relationship. Data showed that dmLT enhanced systemic antibody responses to all seven antigens (CFA/I, CS1-CS6) targeted by MEFA in a dose-dependent way. The adjuvant effect of dmLT on the MEFA construct plateaued at a dose of 0.1 µg. Results also indicated that dmLT is an effective parenteral adjuvant when given by the SC route with the ETEC adhesin MEFA vaccine and that antibody enhancement was achieved with relatively low doses. These observations suggest the potential usefulness of dmLT for parenteral ETEC vaccine candidates and also perhaps for vaccines against other pathogens.

A novel Carcinoembryonic Antigen (CEA)-Targeted Trimeric Immunotoxin shows significantly enhanced Antitumor Activity in Human Colorectal Cancer Xenografts

Immunotoxins are chimeric molecules, which combine antibody specificity to recognize and bind with high-affinity tumor-associated antigens (TAA) with the potency of the enzymatic activity of a toxin, in order to induce the death of target cells. Current immunotoxins present some limitations for cancer therapy, driving the need to develop new prototypes with optimized properties. Herein we describe the production, purification and characterization of two new immunotoxins based on the gene fusion of the anti-carcinoembryonic antigen (CEA) single-chain variable fragment (scFv) antibody MFE23 to α-sarcin, a potent fungal ribotoxin. One construct corresponds to a conventional monomeric single-chain immunotoxin design (IMTXCEAαS), while the other one takes advantage of the trimerbody technology and exhibits a novel trimeric format (IMTXTRICEAαS) with enhanced properties compared with their monomeric counterparts, including size, functional affinity and biodistribution, which endow them with an improved tumor targeting capacity. Our results show the highly specific cytotoxic activity of both immunotoxins in vitro, which was enhanced in the trimeric format compared to the monomeric version. Moreover, the trimeric immunotoxin also exhibited superior antitumor activity in vivo in mice bearing human colorectal cancer xenografts. Therefore, trimeric immunotoxins represent a further step in the development of next-generation therapeutic immunotoxins.

Delivery of Antigen to CD8+ Dendritic Cells by Fusing Antigen With Formyl Peptide Receptor-Like 1 Inhibitor Protein Induces Antitumor Immunity.

A major challenge for vaccine development is targeting antigens to dendritic cells (DCs) in vivo, enabling cross-presentation, and inducing the memory responses. Fcγ receptors (FcγRs) are expressed on many cell types including DCs. Therefore, targeting of antigen to DCs via FcγRs is an attractive strategy for vaccine development. This study employ formyl peptide receptor-like 1 inhibitory protein (FLIPr), an FcγR binding protein secreted by Staphylococcus aureus, to deliver antigen to DCs. Our results show that FLIPr is a competent vehicle in delivering antigen to CD8+ DCs for induction of potent immunities without extra adjuvant formulation. Fusion antigen with FLIPr enables effective antigen presentation on both MHC class II and class I to induce memory T cell responses. Altogether, using FLIPr as an antigen delivery vector has great potential to apply antigens for cancer immunotherapy as well as other infectious disease vaccines.

CD4+ T Cells Drive Lung Disease Enhancement Induced by Immunization with Suboptimal Doses of Respiratory Syncytial Virus Fusion Protein in the Mouse Model.

Respiratory syncytial virus (RSV) infection of seronegative children previously immunized with formalin-inactivated (FI) RSV has been associated with serious enhanced respiratory disease (ERD). The phenomenon was reproduced in the cotton rat and the mouse, and both preclinical models have been routinely used to evaluate the safety of new RSV vaccine candidates. More recently, we demonstrated that immunizations with suboptimal doses of the RSV fusion (F) antigen, in its post- or prefusion conformation, and in the presence of a Th1-biasing adjuvant, unexpectedly led to ERD in the cotton rat model. To assess if those observations are specific to the cotton rat and to elucidate the mechanism by which vaccination with low antigen doses can drive ERD post-RSV challenge, we evaluated RSV post-F antigen dose de-escalation in BALB/c mice in the presence of a Th1-biasing adjuvant. While decreasing antigen doses, we observed an increase in lung inflammation associated with an upregulation of proinflammatory cytokines. The amplitude of the lung histopathology was comparable to that of FI-RSV-induced ERD, confirming the observations made in the cotton rat. Importantly, depletion of CD4+ T cells prior to viral challenge completely abrogated ERD, preventing proinflammatory cytokine upregulation and the infiltration of T cells, neutrophils, eosinophils, and macrophages into the lung. Overall, low-antigen-dose-induced ERD resembles FI-RSV-induced ERD, except that the former appears in the absence of detectable levels of viral replication and in the context of a Th1-biased immune response. Taken together, our observations reinforce the recent concept that vaccines developed for RSV-naïve individuals should be systematically tested under suboptimal dosing conditions.IMPORTANCE RSV poses a significant health care burden and is the leading cause of serious lower-respiratory-tract infections in young children. A formalin-inactivated RSV vaccine developed in the 1960s not only showed a complete lack of efficacy against RSV infection but also induced severe lung disease enhancement in vaccinated children. Since then, establishing safety in preclinical models has been one of the major challenges to RSV vaccine development. We recently observed in the cotton rat model that suboptimal immunizations with RSV fusion protein could induce lung disease enhancement. In the present study, we extended suboptimal dosing evaluation to the mouse model. We confirmed the induction of lung disease enhancement by vaccinations with low antigen doses and dissected the associated immune mechanisms. Our results stress the need to evaluate suboptimal dosing for any new RSV vaccine candidate developed for seronegative infants.

Inactivated Lactobacillus plantarum Carrying a Surface-Displayed Ag85B-ESAT-6 Fusion Antigen as a Booster Vaccine Against Mycobacterium tuberculosis Infection.

Vaccination is considered the most effective strategy for controlling tuberculosis (TB). The existing vaccine, the Bacille Calmette-Guérin (BCG), although partially protective, has a number of limitations. Therefore, there is a need for developing new TB vaccines and several strategies are currently exploited including the use of viral and bacterial delivery vectors. We have previously shown that Lactobacillus plantarum (Lp) producing Ag85B and ESAT-6 antigens fused to a dendritic cell-targeting peptide (referred to as Lp_DC) induced specific immune responses in mice. Here, we analyzed the ability of two Lp-based vaccines, Lp_DC and Lp_HBD (in which the DC-binding peptide was replaced by an HBD-domain directing the antigen to non-phagocytic cells) to activate antigen-presenting cells, induce specific immunity and protect mice from Mycobacterium tuberculosis infection. We tested two strategies: (i) Lp as BCG boosting vaccine (a heterologous regimen comprising parenteral BCG immunization followed by intranasal Lp boost), and (ii) Lp as primary vaccine (a homologous regimen including subcutaneous priming followed by intranasal boost). The results showed that both Lp constructs applied as a BCG boost induced specific cellular immunity, manifested in T cell proliferation, antigen-specific IFN-γ responses and multifunctional T cells phenotypes. More importantly, intranasal boost with Lp_DC or Lp_HBD enhanced protection offered by BCG, as shown by reduced M. tuberculosis counts in lungs. These findings suggest that Lp constructs could be developed as a potential mucosal vaccine platform against mycobacterial infections.

Improving Cross-Protection against Influenza Virus Using Recombinant Vaccinia Vaccine Expressing NP and M2 Ectodomain Tandem Repeats.

Conventional influenza vaccines need to be designed and manufactured yearly. However, they occasionally provide poor protection owing to antigenic mismatch. Hence, there is an urgent need to develop universal vaccines against influenza virus. Using nucleoprotein (NP) and extracellular domain of matrix protein 2 (M2e) genes from the influenza A virus A/Beijing/30/95 (H3N2), we constructed four recombinant vaccinia virus-based influenza vaccines carrying NP fused with one or four copies of M2e genes in different orders. The recombinant vaccinia viruses were used to immunize BALB/C mice. Humoral and cellular responses were measured, and then the immunized mice were challenged with the influenza A virus A/Puerto Rico/8/34 (PR8). NP-specific humoral response was elicited in mice immunized with recombinant vaccinia viruses carrying full-length NP, while robust M2e-specific humoral response was elicited only in the mice immunized with recombinant vaccinia viruses carrying multiple copies of M2e. All recombinant viruses elicited NP- and M2e-specific cellular immune responses in mice. Only immunization with RVJ-4M2eNP induced remarkably higher levels of IL-2 and IL-10 cytokines specific to M2e. Furthermore, RVJ-4M2eNP immunization provided the highest cross-protection in mice challenged with 20 MLD50 of PR8. Therefore, the cross-protection potentially correlates with both NP and M2e-specific humoral and cellular immune responses induced by RVJ-4M2eNP, which expresses a fusion antigen of full-length NP preceded by four M2e repeats. These results suggest that the rational fusion of NP and multiple M2e antigens is critical toward inducing protective immune responses, and the 4M2eNP fusion antigen may be employed to develop a universal influenza vaccine.Electronic supplementary materialThe online version of this article (10.1007/s12250-019-00138-9) contains supplementary material, which is available to authorized users.

Bacterial Outer Membrane Vesicles (OMVs)-based Dual Vaccine for Influenza A H1N1 Virus and MERS-CoV.

Vaccination is the most functional medical intervention to prophylactically control severe diseases caused by human-to-human or animal-to-human transmissible viral pathogens. Annually, seasonal influenza epidemics attack human populations leading to 290–650 thousand deaths/year worldwide. Recently, a novel Middle East Respiratory Syndrome Coronavirus emerged. Together, those two viruses present a significant public health burden in areas where they circulate. Herein, we generated a bacterial outer membrane vesicles (OMVs)-based vaccine presenting the antigenic stable chimeric fusion protein of the H1-type haemagglutinin (HA) of the pandemic influenza A virus (H1N1) strain from 2009 (H1N1pdm09) and the receptor binding domain (RBD) of the Middle East Respiratory Syndrome Coronavirus (MERS-CoV) (OMVs-H1/RBD). Our results showed that the chimeric antigen could induce specific neutralizing antibodies against both strains leading to protection of immunized mice against H1N1pdm09 and efficient neutralization of MERS-CoV. This study demonstrate that OMVs-based vaccines presenting viral antigens provide a safe and reliable approach to protect against two different viral infections.

Mapping the Neutralizing Epitopes of Enterotoxigenic Escherichia coli K88 (F4) Fimbrial Adhesin and Major Subunit FaeG.

Enterotoxigenic Escherichia coli (ETEC) strains that produce immunologically heterogeneous fimbriae and enterotoxins are the primary cause of neonatal diarrhea and postweaning diarrhea in young pigs. A multivalent vaccine inducing protective immunity against ideally all ETEC fimbriae and enterotoxins could be effective against diarrhea in young pigs. However, developing a vaccine to broadly protect against various ETEC virulence determinants has proven challenging. Recently developed structure- and epitope-based multiepitope fusion antigen (MEFA) technology that presents neutralizing epitopes of various virulence determinants at a backbone immunogen and that mimics epitope native immunogenicity suggests the feasibility of developing multivalent vaccines. With neutralizing epitopes from ETEC fimbria F18 and enterotoxins being identified, it becomes urgent to identify protective epitopes of K88 (F4) fimbriae, which play a major role in pig neonatal and postweaning diarrhea. In this study, we identified B-cell immunodominant epitopes in silico from the K88ac fimbrial major subunit (also adhesin) FaeG and embedded each epitope in a heterogeneous carrier for epitope fusions. We then immunized mice with each epitope fusion protein and examined epitope antigenicity and also neutralizing activities of epitope-induced antibodies. Data showed that while all nine FaeG epitope fusions induced antibodies to K88ac fimbria, anti-K88 IgG antibodies derived from epitopes MTGDFNGSVD (ep1), LNDLTNGGTK (ep2), GRTKEAFATP (ep3), ELRKPDGGTN (ep4), PMKNAGGTKVGAVKVN (ep5), and RENMEYTDGT (ep8) significantly inhibited adherence of K88ac fimbrial bacteria to porcine intestinal cell line IPEC-J2, indicating that these peptides were the neutralizing epitopes of K88ac fimbrial major subunit FaeG and suggesting the future application of FaeG epitopes in ETEC vaccine development.IMPORTANCE Enterotoxigenic Escherichia coli (ETEC) strains producing K88ac fimbriae and enterotoxins are a major cause of porcine neonatal diarrhea and postweaning diarrhea in the United States. Currently, there is no vaccine to induce broadly protective antiadhesin and antitoxin immunity against ETEC-associated diarrhea. To develop a broadly effective ETEC vaccine, we need to target the most important if not all ETEC virulence determinants. While conventional vaccinology approaches encounter difficulties at integrating or including heterogeneous ETEC fimbria and toxin antigens into a vaccine product, multiepitope fusion antigen (MEFA) structural vaccinology provides a new platform to combine neutralizing antigenic elements or epitopes from various heterogeneous virulence factors for broad immunity and protection. Identification of the neutralizing epitopes of K88ac fimbria from this study added the last antigens to an MEFA-based multivalent vaccine against ETEC-associated diarrhea in pigs. An effective vaccine against pig diarrhea can significantly improve swine health and well-being and reduce economic losses to the swine industry worldwide.

Construction and expression of a single-chain variable fragment antibody against chicken DEC 205 for targeting the bacterial expressed hemagglutinin-neuraminidase of Newcastle disease virus

Targeting antigens to endocytic receptors on the surface of dendritic cells is a new strategy for increasing the adaptive immune response. The objective of the current study was the construction and bacterial expression of a recombinant antibody single-chain fragment variable (ScFv) directed against chicken DEC 205, an endocytic receptor, for use in the genetic fusion of antigens. In particular, we use as antigen the hemagglutinin-neuraminidase (HN) of Newcastle disease virus. Our results show that inoculation of chickens with HN genetically fused to the ScFv anti-DEC 205 induced an evidently higher immune response against HN, in contrast to inoculation with unconjugated HN. In addition, neutralizing antibodies against Newcastle disease virus were detected only in the serum from chickens immunized with HN fused to ScFv anti-DEC 205. Inoculated fused antigens to ScFv against endocytic receptor DEC 205 resulted in a greater antibody-specific anti-HN production compared with antigens applied alone. The results of this study show that the strategy described here has the potential to be used in the development of more effective vaccines against infectious diseases in chickens.

CD40 ligand (CD40L) multi-trimer antigen fusion protein (MagaVax) as a vaccine design for high-level CD8+ T cell responses

Abstract Dendritic cells (DCs) can take up antigen via a number of routes. Of these, several studies have shown antigen uptake via the CD40 receptor generates some of the strongest CD8+ T cell responses. At the same time, stimulation through CD40 activates DCs for cross-presentation and heightened antigen presentation. Consequently, it is desirable to combine the CD40 targeting of antigen and a CD40 stimulant into a single vaccine formulation. When the CD40 stimulant is CD40 ligand (CD40L, CD154), it is important to use a multi-trimer complex that clusters CD40 in the DC membrane, thereby engaging downstream signaling pathways. Such a multi-trimer form of CD40L can be provided by fusing the extracellular domain of CD40L with the body of surfactant protein D (SPD), a self-assembling 12-chain soluble molecule with 4 trimeric “arms.” When an antigen sequence is included within the SPD arm, the entire protein complex is termed “Multi-trimer AntiGen Adjuvant Vaccine (Vax)” or “MagaVax.” Studies in mice have shown that MagaVax elicits very high levels of antigen-specific CD8+ T cells with high TCR avidity and functional activity. MagaVax can be effectively delivered as a DNA vaccine i.m. and even more effectively using an adenoviral (Ad5) vector. For a MagaVax encoding HIV-1 Gag, vaccination with Ad5-MagaVax-Gag led to complete protection (“sterilizing immunity”) from Vaccinia-Gag challenge (i.e., 7 log reduction of tissue virus). Thus far, MagaVax formulations have been made for HIV-1 Gag, Ebola, malaria, hepatitis B virus (HBV), and tumor-specific neoantigens. In all of these cases, there may be a clinical benefit for a vaccine that elicits very strong CD8+ T cell responses.

Combination of Synthetic Long Peptides and XCL1 Fusion Proteins Results in Superior Tumor Control

Cross-presenting Xcr1+CD8α DCs are attractive APCs to target for therapeutic cancer vaccines, as they are able to take up and process antigen from dying tumor cells for their MHCI-restricted presentation to CD8 T cells. To this aim, we developed fusion proteins made of the Xcr1 ligand Xcl1 fused to an OVA synthetic long peptide (SLP) and IgG1 Fc fragment. We demonstrated the specific binding and uptake of the Xcl1 fusion proteins by Xcr1+ DCs. Most importantly, their potent adjuvant effect on the H-2Kb/OVA specific T cell response was associated with a sustained tumor control even against the poorly immunogenic B16-OVA melanoma tumor. The increased tumor protection correlated with higher tumor infiltration of antigen-specific CD8+ T cells, increased IFNγ production and degranulation potential. Altogether, these results demonstrate that therapeutic cancer vaccines may be greatly improved by the combination of SLP antigen and Xcl1 fusion proteins.

Dual-Isotope SPECT/CT Imaging of the Tuberculosis Subunit Vaccine H56/CAF01: Induction of Strong Systemic and Mucosal IgA and T-Cell Responses in Mice Upon Subcutaneous Prime and Intrapulmonary Boost Immunization.

Pulmonary tuberculosis (TB), which is caused by Mycobacterium tuberculosis (Mtb), remains a global pandemic, despite the widespread use of the parenteral live attenuated Bacillus Calmette–Guérin (BCG) vaccine during the past decades. Mucosal administration of next generation TB vaccines has great potential, but developing a safe and efficacious mucosal vaccine is challenging. Hence, understanding the in vivo biodistribution and pharmacokinetics of mucosal vaccines is essential for shaping the desired immune response and for optimal spatiotemporal targeting of the appropriate effector cells in the lungs. A subunit vaccine consisting of the fusion antigen H56 (Ag85B-ESAT-6-Rv2660) and the liposome-based cationic adjuvant formulation (CAF01) confers efficient protection in preclinical animal models. In this study, we devise a novel immunization strategy for the H56/CAF01 vaccine, which comply with the intrapulmonary (i.pulmon.) route of immunization. We also describe a novel dual-isotope (111In/67Ga) radiolabeling approach, which enables simultaneous non-invasive and longitudinal SPECT/CT imaging and quantification of H56 and CAF01 upon parenteral prime and/or i.pulmon. boost immunization. Our results demonstrate that the vaccine is distributed evenly in the lungs, and there are pronounced differences in the pharmacokinetics of H56 and CAF01. We provide convincing evidence that the H56/CAF01 vaccine is not only well-tolerated when administered to the respiratory tract, but it also induces strong lung mucosal and systemic IgA and polyfunctional Th1 and Th17 responses after parenteral prime and i.pulmon. boost immunization. The study furthermore evaluate the application of SPECT/CT imaging for the investigation of vaccine biodistribution after parenteral and i.pulmon. immunization of mice.

Establishment and preliminary application of optimal cut-off value of anti-multi-epitope fusion antigen IgG antibody of Mycobacterium tuberculosis

Establishment and preliminary application of optimal cut-off value of anti-multi-epitope fusion antigen IgG antibody of Mycobacterium tuberculosis

Heterologous Immunization With Defined RNA and Subunit Vaccines Enhances T Cell Responses That Protect Against Leishmania donovani.

The rapid generation of strong T cell responses is highly desirable and viral vectors can have potent CD8+ T cell-inducing activity. Immunity to leishmaniasis requires selective T cell responses, with immunization schemes that raise either CD4 or CD8 T cell responses being protective in small animal models. We have defined the leishmaniasis vaccine candidate recombinant fusion antigens, LEISH-F2 and LEISH-F3+, that when formulated in a stable emulsion with a Toll-like receptor (TLR) 4 agonist, induce protective CD4+ T cell responses in animal models as well as providing therapeutic efficacy in canine leishmaniasis and in clinical trials in leishmaniasis patients. We used the genetic sequences of these validated vaccine antigens to design RNA vaccine constructs. Immunization of mice with the RNA replicons induced potent, local innate responses that were surprisingly independent of TLR7 and activated antigen-presenting cells (APC) to prime for extremely potent antigen-specific T helper 1 type responses upon heterologous boosting with either of the subunit vaccines (recombinant antigen with second generation glucopyranosyl lipid A in stable oil-in-water emulsion; SLA-SE). Inclusion of RNA in the immunization schedule also generated MHCI-restricted T cell responses. Immunization with LEISH-F2-expressing RNA vaccine followed later by subunit vaccine afforded protection against challenge with Leishmania donovani. Together, these data indicate the utility of heterologous prime-boost immunization schemes for the induction of potent antigen-specific CD4 and CD8 T cell responses for protection against intracellular pathogens.

INTERFERON GAMMA AND INTERLEUKIN-10 LEVELS IN PBMC OF ACTIVE AND LATENT TUBERCULOSIS PATIENTS AS WELL AS HEALTHY INDIVIDUALS

Tuberculosis (TB), an infectious disease caused by a Mycobacterium tuberculosis, is still a health problem in Indonesia, and the world. One of the failures to control the TB epidemic is due to the lack of effective vaccines available today. Protective immune responses to M.tuberculosis are dominated by cellular immunity and less by humoral immunity. IFN-γ, and IL-10 play a role in the protection of against M.tuberculosis, and the pathogenesis of TB. Fusion antigen ESAT-6-CFP-10 has a strong antigenicity to T cells and stimulates specific cellular immune responses, thereby providing benefit in immune responses that are protective against M.tuberculosis infection. The aimed of this study was to know the difference betwen IFN-γ, and IL-10 levels on PBMC culture of active TB, latent TB, and healthy people after ESAT-6-CFP-10 fusion antigen stimulation. This study used an in vitro of quasi experimental design in PBMC cultures of active TB, latent TB, and healthy people groups stimulated by ESAT-6-CFP-10 antigen fusion Mycobacterium tuberculosis. IFN-γ, and IL-10 levels were measured by ELISA method. The results were analyzed by one-way ANOVA. The mean levels of IFN-γ post-stimulation of ESAT-6-CFP-10 fusion antigens did not differ (p=0.359) in the active pulmonary TB group (0.07 - 2114), latent TB (6.84 - 1381) and healthy people 1.88 - 1807.70), as well as the mean levels of IL-10 (p=0.712) in the active pulmonary TB (16.70 - 328.80), latent TB (29.70 - 323.60 ) and healthy people (31.30 - 958). There were no significant differences in levels of IFN-γ and IL-10 in active TB, latent TB, and healthy people after stimulation by fusion antigen ESAT-6-CFP-10.

Recent Developments in Recombinant Protein-Based Dengue Vaccines.

Recombinant proteins are gaining enormous importance these days due to their wide application as biopharmaceutical products and proven safety record. Various recombinant proteins of therapeutic and prophylactic importance have been successfully produced in microbial and higher expression host systems. Since there is no specific antiviral therapy available against dengue, the prevention by vaccination is the mainstay in reducing the disease burden. Therefore, efficacious vaccines are needed to control the spread of dengue worldwide. Dengue is an emerging viral disease caused by any of dengue virus 1–4 serotypes that affects the human population around the globe. Dengue virus is a single stranded RNA virus encoding three structural proteins (capsid protein, pre-membrane protein, and envelope protein) and seven non-structural proteins (NS1, NS2a, NS2b, NS3, NS4a, NS4b, NS5). As the only licensed dengue vaccine (Dengvaxia) is unable to confer balanced protection against all the serotypes, therefore various approaches for development of dengue vaccines including tetravalent live attenuated, inactivated, plasmid DNA, virus-vectored, virus-like particles, and recombinant subunit vaccines are being explored. These candidates are at different stages of vaccine development and have their own merits and demerits. The promising subunit vaccines are mainly based on envelope or its domain and non-structural proteins of dengue virus. These proteins have been produced in different hosts and are being investigated for development of a successful dengue vaccine. Novel immunogens have been designed employing various strategies like protein engineering and fusion of antigen with various immunostimulatory motif to work as self-adjuvant. Moreover, recombinant proteins can be formulated with novel adjuvants to enhance the immunogenicity and thus conferring better protection to the vaccinees. With the advent of newer and safer host systems, these recombinant proteins can be produced in a cost effective manner at large scale for vaccine studies. In this review, we summarize recent developments in recombinant protein based dengue vaccines that could lead to a good number of efficacious vaccine candidates for future human use and ultimately alternative dengue vaccine candidates.

A polymeric immunoglobulin-antigen fusion protein strategy for enhancing vaccine immunogenicity.

SummaryIn this study, a strategy based on polymeric immunoglobulin G scaffolds (PIGS) was used to produce a vaccine candidate for Mycobacterium tuberculosis. A genetic fusion construct comprising genes encoding the mycobacterial Ag85B antigen, an immunoglobulin γ‐chain fragment and the tailpiece from immunoglobulin μ chain was engineered. Expression was attempted in Chinese Hamster Ovary (CHO) cells and in Nicotiana benthamiana. The recombinant protein assembled into polymeric structures (TB‐PIGS) in N. benthamiana, similar in size to polymeric IgM. These complexes were subsequently shown to bind to the complement protein C1q and FcγRs with increased affinity. Modification of the N‐glycans linked to TB‐PIGS by removal of xylose and fucose residues that are normally found in plant glycosylated proteins also resulted in increased affinity for low‐affinity FcγRs. Immunization studies in mice indicated that TB‐PIGS are highly immunogenic with and without adjuvant. However, they did not improve protective efficacy in mice against challenge with M. tuberculosis compared to conventional vaccination with BCG, suggesting that additional or alternative antigens may be needed to protect against this disease. Nevertheless, these results establish a novel platform for producing polymeric antigen‐IgG γ‐chain molecules with inherent functional characteristics that are desirable in vaccines.