Immunogenicity Studies In Mice Research Articles

Rhabdovirus-based vaccine platforms against henipaviruses.

The emerging zoonotic pathogens Hendra virus (HeV) and Nipah virus (NiV) are in the genus Henipavirus in the family Paramyxoviridae. HeV and NiV infections can be highly fatal to humans and livestock. The goal of this study was to develop candidate vaccines against henipaviruses utilizing two well-established rhabdoviral vaccine vector platforms, recombinant rabies virus (RABV) and recombinant vesicular stomatitis virus (VSV), expressing either the codon-optimized or the wild-type (wt) HeV glycoprotein (G) gene. The RABV vector expressing the codon-optimized HeV G showed a 2- to 3-fold increase in incorporation compared to the RABV vector expressing wt HeV G. There was no significant difference in HeV G incorporation in the VSV vectors expressing either wt or codon-optimized HeV G. Mice inoculated intranasally with any of these live recombinant viruses showed no signs of disease, including weight loss, indicating that HeV G expression and incorporation did not increase the neurotropism of the vaccine vectors. To test the immunogenicity of the vaccine candidates, we immunized mice intramuscularly with either one dose of the live vaccines or 3 doses of 10 μg chemically inactivated viral particles. Increased codon-optimized HeV G incorporation into RABV virions resulted in higher antibody titers against HeV G compared to inactivated RABV virions expressing wt HeV G. The live VSV vectors induced more HeV G-specific antibodies as well as higher levels of HeV neutralizing antibodies than the RABV vectors. In the case of killed particles, HeV neutralizing serum titers were very similar between the two platforms. These results indicated that killed RABV with codon-optimized HeV G should be the vector of choice as a dual vaccine in areas where rabies is endemic. Scientists have been tracking two new viruses carried by the Pteropid fruit bats: Hendra virus (HeV) and Nipah virus (NiV). Both viruses can be fatal to humans and also pose a serious risk to domestic animals. A recent escalation in the frequency of outbreaks has increased the need for a vaccine that prevents HeV and NiV infections. In this study, we performed an extensive comparison of live and killed particles of two recombinant rhabdoviral vectors, rabies virus and vesicular stomatitis virus (VSV), expressing wild-type or codon-optimized HeV glycoprotein, with the goal of developing a candidate vaccine against HeV. Based on our data from the presented mouse immunogenicity studies, we conclude that a killed RABV vaccine would be highly effective against HeV infections and would make an excellent vaccine candidate in areas where both RABV and henipaviruses pose a threat to human health.

Antigenicity studies in humans and immunogenicity studies in mice: an MSP1P subdomain as a candidate for malaria vaccine development

The newly identified GPI-anchored Plasmodium vivax merozoite surface protein 1 paralog (MSP1P) has a highly antigenic C-terminus that binds erythrocytes. To characterize the antigenicity and immunogenicity of two regions (PvMSP1P-19 and -33) of the highly conserved C-terminus of MSP1P relative to PvMSP1-19, 30 P. vivax malaria-infected patients and two groups of mice (immunized with PvMSP1P-19 or -33) were tested for IgG subclass antibodies against PvMSP1P-19 and -33 antigens. In the patients infected with P. vivax, IgG1 and IgG3 levels were significantly higher than those levels in healthy individuals, and were the predominant response to the two C-terminal fragments of PvMSP1P (p < 0.05). In mice immunized with PvMSP1P-19, IgG1 levels were the highest while IgG2b levels were similar to IgG1 levels. The levels of Th1 cytokines in mice immunized with PvMSP1P-19 or -33 were significantly higher than those in mice immunized with PvMSP1-19 (p < 0.05). Our results indicate that: (i) IgG1 and IgG3 (IgG2b in mice) are predominant IgG subclasses in both patients infected with P. vivax and mice immunized with PvMSP1P-19 or -33; (ii) the C-terminus of MSP1P induces a Th1-cytokine response. This immune profiling study provides evidence that MSP1P may be a potential candidate for vivax vaccine.

Mutations in Subunit Interface and B-cell Epitopes Improve Antileukemic Activities of Escherichia coli Asparaginase-II: EVALUATION OF IMMUNOGENICITY IN MICE

L-Asparaginase-II from Escherichia coli (EcA) is a central component in the treatment of acute lymphoblastic leukemia (ALL). However, the therapeutic efficacy of EcA is limited due to immunogenicity and a short half-life in the patient. Here, we performed rational mutagenesis to obtain EcA variants with a potential to improve ALL treatment. Several variants, especially W66Y and Y176F, killed the ALL cells more efficiently than did wild-type EcA (WT-EcA), although nonleukemic peripheral blood monocytes were not affected. Several assays, including Western blotting, annexin-V/propidium iodide binding, comet, and micronuclei assays, showed that the reduction in viability of leukemic cells is due to the increase in caspase-3, cytochrome c release, poly(ADP-ribose) polymerase activation, down-regulation of anti-apoptotic protein Bcl-XL, an arrest of the cell cycle at the G0/G1 phase, and eventually apoptosis. Both W66Y and Y176F induced significantly more apoptosis in lymphocytes derived from ALL patients. In addition, Y176F and Y176S exhibited greatly decreased glutaminase activity, whereas K288S/Y176F, a variant mutated in one of the immunodominant epitopes, showed reduced antigenicity. Further in vivo immunogenicity studies in mice showed that K288S/Y176F was 10-fold less immunogenic as compared with WT-EcA. Moreover, sera obtained from WT-EcA immunized mice and ALL patients who were given asparaginase therapy for several weeks recognized the K288S/Y176F mutant significantly less than the WT-EcA. Further mechanistic studies revealed that W66Y, Y176F, and K288S/Y176F rapidly depleted asparagine and also down-regulated the transcription of asparagine synthetase as compared with WT-EcA. These highly desirable attributes of these variants could significantly advance asparaginase therapy of leukemia in the future.

Improved storage stability and immunogenicity of hepatitis B vaccine after spray-freeze drying in presence of sugars

The current hepatitis B vaccines need to be stored and transported under refrigerated conditions (2–8°C). This dependence on a cold-chain is highly challenging in areas where hepatitis B virus infections are endemic. To decrease the cold-chain dependency, powder formulations of the hepatitis B surface antigen (HBsAg) without aluminum were prepared by spray-freeze drying in the presence of either inulin or a combination of dextran and trehalose. The stability of HBsAg in the amorphous powder formulations was strongly improved during storage both at room temperature and at an elevated temperature (60°C), compared to a liquid plain and an aluminum hydroxide adjuvanted HBsAg formulation. Immunogenicity studies in mice showed that reconstituted powder formulations induced higher IgG immune responses after intramuscular administration than those induced after administration of unprocessed plain antigen. Although the immune response was not as high as after administration of aluminum adjuvanted HBsAg, the immune response to the reconstituted vaccines shifted towards a more balanced Th1/Th2 response compared to the aluminum containing HBsAg formulation.

Immunogenicity studies of bivalent inactivated virions of EV71/CVA16 formulated with submicron emulsion systems.

We assessed two strategies for preparing candidate vaccines against hand, foot, and mouth disease (HFMD) caused mainly by infections of enterovirus (EV) 71 and coxsackievirus (CV) A16. We firstly design and optimize the potency of adjuvant combinations of emulsion-based delivery systems, using EV71 candidate vaccine as a model. We then perform immunogenicity studies in mice of EV71/CVA16 antigen combinations formulated with PELC/CpG. A single dose of inactivated EV71 virion (0.2 μg) emulsified in submicron particles was found (i) to induce potent antigen-specific neutralizing antibody responses and (ii) consistently to elicit broad antibody responses against EV71 neutralization epitopes. A single dose immunogenicity study of bivalent activated EV71/CVA16 virion formulated with either Alum or PELC/CpG adjuvant showed that CVA16 antigen failed to elicit CVA16 neutralizing antibody responses and did not affect EV71-specific neutralizing antibody responses. A boosting dose of emulsified EV71/CVA16 bivalent vaccine candidate was found to be necessary to achieve high seroconversion of CVA16-specific neutralizing antibody responses. The current results are important for the design and development of prophylactic vaccines against HFMD and other emerging infectious diseases.

The Development of an Experimental Multiple Serogroups Vaccine for Neisseria meningitidis

A native outer membrane vesicles (NOMV) vaccine was developed from three antigenically diverse strains of Neisseria meningitidis that express the L1,8, L2, and L3,7 lipooligosaccharide (LOS) immunotypes, and whose synX, and lpxL1 genes were deleted.. Immunogenicity studies in mice showed that the vaccine induced bactericidal antibody against serogroups B, C, W, Y and X N. meningitidis strains. However, this experimental NOMV vaccine was not effective against serogroup A N. meningitidis strains. N. meningitidis capsular polysaccharide (PS) from serogroups A, C, W and Y were effective at inducing bactericidal antibody when conjugated to either tetanus toxoid or the fHbp1-fHbp2 fusion protein fHbp(1+2). The combination of the NOMV vaccine and the N. meningitidis serogroup A capsular polysaccharide (MAPS) protein conjugate was capable of inducing bactericidal antibodies against a limited number of N. meningitidis strains from serogroups A, B, C, W, Y and X tested in this study.

Immunological Evaluation and Comparison of Different EV71 Vaccine Candidates

Enterovirus 71 (EV71) and coxsackievirus A16 (CVA16) are major causative agents of hand, foot, and mouth diseases (HFMDs), and EV71 is now recognized as an emerging neurotropic virus in Asia. Effective medications and/or prophylactic vaccines against HFMD are not available. The current results from mouse immunogenicity studies using in-house standardized RD cell virus neutralization assays indicate that (1) VP1 peptide (residues 211–225) formulated with Freund's adjuvant (CFA/IFA) elicited low virus neutralizing antibody response (1/32 titer); (2) recombinant virus-like particles produced from baculovirus formulated with CFA/IFA could elicit good virus neutralization titer (1/160); (3) individual recombinant EV71 antigens (VP1, VP2, and VP3) formulated with CFA/IFA, only VP1 elicited antibody response with 1/128 virus neutralization titer; and (4) the formalin-inactivated EV71 formulated in alum elicited antibodies that cross-neutralized different EV71 genotypes (1/640), but failed to neutralize CVA16. In contrast, rabbits antisera could cross-neutralize strongly against different genotypes of EV71 but weakly against CVA16, with average titers 1/6400 and 1/32, respectively. The VP1 amino acid sequence dissimilarity between CVA16 and EV71 could partially explain why mouse antibodies failed to cross-neutralize CVA16. Therefore, the best formulation for producing cost-effective HFMD vaccine is a combination of formalin-inactivated EV71 and CAV16 virions.

Recombinant BCG coexpressing Ag85B, CFP10, and interleukin‐12 induces multifunctional Th1 and memory T cells in mice

Mycobacterium tuberculosis (MTB) continues to be a leading cause of human deaths due to an infectious agent. Current efforts are focused on making better TB vaccines. We describe the generation and immunological characterization of recombinant BCG (rBCG). This rBCG was generated by incorporating an expression plasmid encoding two mycobacterial antigens (Ag85B and CFP10) and human interleukin (IL)-12 into a BCG strain. Immunogenicity studies in mice showed that rBCG coexpressing Ag85B, CFP10, and IL-12 (rBCG::Ag85B-CFP10-IL-12) induces a robust immune response in mice. The rBCG vaccine promotes a T-cell response against MTB that is characterized by a high proportion of polyfunctional and memory T cells in spleen and lung. Our results showed strong immunogenicity and mycobacterial growth inhibition of rBCG::Ag85B-CFP10 plus IL-12 than that of BCG vaccine.

An experimental outer membrane vesicle vaccine from N. meningitidis serogroup B strains that induces serum bactericidal activity to multiple serogroups

A trivalent native outer membrane vesicle vaccine that has potential to provide broad based protection against Neisseria meningitidis serogroup B strains has been developed. Preliminary immunogenicity studies in mice showed that the vaccine was capable of inducing an effective broad based bactericidal antibody response against N. meningitidis serogroup B strains. These findings in mice have been repeated with a cGMP trivalent NOMV vaccine and extended to show that the bactericidal antibody response induced by the vaccine in mice is effective against strains belonging to serogroups C, Y, W135, X, and NadA-expressing serogroup A strains. Taken together these results suggest that this experimental vaccine may provide protection against both serogroup B and non-serogroup B N. meningitidis strains.

Purification and Characterization of Enterovirus 71 Viral Particles Produced from Vero Cells Grown in a Serum-Free Microcarrier Bioreactor System

BackgroundEnterovirus 71 (EV71) infections manifest most commonly as a childhood exanthema known as hand-foot-and-mouth disease (HFMD) and can cause neurological disease during acute infection.Principal FindingIn this study, we describe the production, purification and characterization of EV71 virus produced from Vero cells grown in a five-liter serum-free bioreactor system containing 5 g/L Cytodex 1 microcarrier. The viral titer was >106 TCID50/mL by 6 days post infection when a MOI of 10−5 was used at the initial infection. Two EV71 virus fractions were separated and detected when the harvested EV71 virus concentrate was purified by sucrose gradient zonal ultracentrifugation. The EV71 viral particles detected in the 24–28% sucrose fractions had an icosahedral structure 30–31 nm in diameter and had low viral infectivity and RNA content. Three major viral proteins (VP0, VP1 and VP3) were observed by SDS-PAGE. The EV71 viral particles detected in the fractions containing 35–38% sucrose were 33–35 nm in size, had high viral infectivity and RNA content, and were composed of four viral proteins (VP1, VP2, VP3 and VP4), as shown by SDS-PAGE analyses. The two virus fractions were formalin-inactivated and induced high virus neutralizing antibody responses in mouse immunogenicity studies. Both mouse antisera recognized the immunodominant linear neutralization epitope of VP1 (residues 211–225).ConclusionThese results provide important information for cell-based EV71 vaccine development, particularly for the preparation of working standards for viral antigen quantification.

Carnauba wax nanoparticles enhance strong systemic and mucosal cellular and humoral immune responses to HIV-gp140 antigen

Induction of humoral responses to HIV at mucosal compartments without inflammation is important for vaccine design. We developed charged wax nanoparticles that efficiently adsorb protein antigens and are internalized by DC in the absence of inflammation. HIV-gp140-adsorbed nanoparticles induced stronger in vitro T-cell proliferation responses than antigen alone. Such responses were greatly enhanced when antigen was co-adsorbed with TLR ligands. Immunogenicity studies in mice showed that intradermal vaccination with HIV-gp140 antigen-adsorbed nanoparticles induced high levels of specific IgG. Importantly, intranasal immunization with HIV-gp140-adsorbed nanoparticles greatly enhanced serum and vaginal IgG and IgA responses. Our results show that HIV-gp140-carrying wax nanoparticles can induce strong cellular/humoral immune responses without inflammation and may be of potential use as effective mucosal adjuvants for HIV vaccine candidates.

Evaluation of yellow fever virus 17D strain as a new vector for HIV-1 vaccine development

The failure to develop an effective vaccine against HIV-1 infection has led the research community to seek new ways of raising qualitatively different antibody and cellular immune responses. Towards this goal, we investigated the yellow fever 17D vaccine strain (YF17D), one of the most effective vaccines ever made, as a platform for HIV-1 vaccine development. A test antigen, HIV-1 p24 (clade B consensus), was inserted near the 5′ end of YF17D, in frame and upstream of the polyprotein (YF-5′/p24), or between the envelope and the first non-structural protein (YF-E/p24/NS1). In vitro characterization of these recombinants indicated that the gene insert was more stable in the context of YF-E/p24/NS1. This was confirmed in immunogenicity studies in mice. CD8+ IFN-γ T-cell responses against p24 were elicited by the YF17D recombinants, as were specific CD4+ T cells expressing IFN-γ and IL-2. A balanced CD4+ and CD8+ T-cell response was notable, as was the polyfunctionality of the responding cells. Finally, the protective efficacy of the YF17D recombinants, particularly YF-E/p24/NS1, in mice challenged with a vaccinia expressing HIV-1 Gag was demonstrated. These results suggest that YF17D warrants serious consideration as a live-attenuated vector for HIV-1 vaccine development.

HLA footprinting reveals a novel protective CD8 + T cell response in Hepatitis C virus infection

vectors have generated great scientific interest in recent years and appear to be superior viral vectors with great potential in vaccine regimes. Their potential use in humans, however, is limited by natural anti-vector immunity to human adenoviruses, but this problem could be largely circumvented by the use of simian adenoviral vaccine vectors. Recent clinical trials have suggested that the simian adenoviral vector AdCh63 is a promising clinical candidate. We have developed vectors (of human and simian origin) and MVA encoding a novel construct based on P. falciparum MSP-1 and have undertaken comparative immunogenicity studies in mice. The antigen, termed ‘PfM128, is based on the five highly conserved blocks from P. falciparum MSP-1, plus both dimorphic alleles of the 42 kDa region. We have used these vectors to map novel murine Tcell epitopes within PfMSP-1. We compare antibody titres and polyfunctional T cell responses when simian adenoviral vector AdCh63 is used to replace AdHu5 in a heterologous prime-boost regime. These data suggest that simian adenoviral vectors have great potential for use in future blood-stage malaria vaccination regimes in humans.

Toward the Rational Design of a Malaria Vaccine Construct Using the MSP3 Family as an Example: Contribution of Antigenicity Studies in Humans

Plasmodium falciparum merozoite surface protein (MSP3) is a main target of protective immunity against malaria that is currently undergoing vaccine development. It was shown recently to belong, together with MSP6, to a new multigene family whose C-terminal regions have a similar organization, contain both homologous and divergent regions, and are highly conserved across isolates. In an attempt to rationally design novel vaccine constructs, we extended the analysis of antigenicity and function of region-specific antibodies, previously performed with MSP3 and MSP6, to the remaining four proteins of the MSP3 family using four recombinant proteins and 24 synthetic peptides. Antibodies to each MSP3 family antigen were found to be highly prevalent among malaria-exposed individuals from the village of Dielmo (Senegal). Each of the 24 peptides was antigenic, defining at least one epitope mimicking that of the native proteins, with a distinct IgG isotype pattern for each, although with an overall predominance of the IgG3 subclass. Human antibodies affinity purified upon each of the 24 peptides exerted an antiparasite antibody-dependent cellular inhibition effect, which in most cases was as strong as that of IgG from protected African adults. The two regions with high homology were found to generate a broad network of cross-reactive antibodies with various avidities. A first multigenic construct was designed using these findings and those from related immunogenicity studies in mice and demonstrated valuable immunological properties. These results indicate that numerous regions from the MSP3 family play a role in protection and provide a rationale for the tailoring of new MSP3-derived malaria vaccines.

Formulation and immunological evaluation of novel vaccine delivery systems based on bioresorbable poly(ethylene glycol)‐block‐poly(lactide‐co‐ε‐caprolactone)

Novel emulsion-type vaccine delivery systems based on the amphiphilic bioresorbable polymer poly(ethylene glycol)-block-poly(lactide-co-epsilon-caprolactone) (PEG-b-PLACL) and selected oils were developed here. Physicochemical characterizations such as stability, a droplet test, microscopic aspects, and in vitro release showed that PEG-b-PLACL-emulsified formulations have several advantages over traditional vaccine adjuvants in that they are stable, reproducible, and homogeneous fine particles with an appropriate size to facilitate the induction of potent immune responses. Different dispersion-type emulsions have provided different release profiles using ovalbumin in model studies. Immunogenicity studies in mice have shown that antigen-specific antibody titers and T-cell proliferative responses, as well as the secretion of IFN-gamma, were significantly enhanced for ovalbumin after formulation with PEG-b-PLACL-based emulsions. These features are of great interest for applications in delivery systems of prophylactic and therapeutic vaccine candidates.

Recombinant virus-like particles as a carrier of B- and T-cell epitopes of hepatitis C virus (HCV)

The major aim of the project was the development of virus-like particles (VLP) displaying B- and T-cell epitopes of hepatitis C virus (HCV) proteins. To this end, hepatitis B virus core (HBc) particles were used as a carrier of HCV epitopes. Fragments of HCV genes encoding core (aa 98) and NS3 (aa 155) proteins were fused to the 3′ terminus of the truncated HBV core gene. All recombinant plasmids led to relatively high levels of expression of chimeric proteins in E. coli, which resulted in the formation of complete “mature” VLP. Chimeric HBc/HCV VLPs were purified by combination of gel filtration and sucrose gradient centrifugation, and used for immunogenicity studies in mice. All variants of hybrid particles induced high humoral and cellular responses to HBcAg. Immunization with the HBc/HCV core particles led to relatively low antibody and T-cell proliferative responses to HCV core epitopes. The HBc/HCV NS3 particles were able to induce high levels of anti-NS3 antibodies in the absence of proliferative responses to HCV epitopes. Thus, the results of the current study have demonstrated the principal possibility of using VLP on the basis of HBcAg for creation of a new type of HCV-specific immunogen.

A novel adenovirus type 6 (Ad6)-based hepatitis C virus vector that overcomes preexisting anti-ad5 immunity and induces potent and broad cellular immune responses in rhesus macaques.

Success in resolving hepatitis C virus (HCV) infection has been correlated to vigorous, multispecific, and sustained CD8(+) T-cell response in humans and chimpanzees. The efficacy of inducing T-cell-mediated immunity by recombinant serotype 5 adenovirus vector has been proven in many animal models of infectious diseases, but its immunogenicity can be negatively influenced by preexisting immunity against the vector itself. To evaluate the less prevalent adenovirus serotype 6 (Ad6) as an alternative vector for and HCV vaccine development, we have generated serotype 5 and 6 adenoviral vectors directing expression of the nonstructural region of HCV (MRKAd5-NSmut and MRKAd6-NSmut). Immunogenicity studies in mice showed that the two vectors induced comparable T-cell responses but that only MRKAd6-NSmut was not suppressed in the presence of anti-Ad5 immunity. In contrast, preexisting anti-Ad5 immunity dramatically blunted the immunogenicity of the serotype 5-based HCV vector. Furthermore, MRKAd6-NSmut showed equivalent potency, breadth, and longevity of HCV-specific T-cell responses in rhesus macaques as the corresponding Ad5-based vector over a wide range of doses and was capable of boosting DNA-primed animals even if administered at low doses. These data support the use of the MRKAd6-NSmut for anti-HCV immunotherapy and, more generally, for the Ad6 serotype as a better genetic vaccine vehicle than Ad5.

Molecular Cloning and Immunologic Reactivity of a Novel Low Molecular Mass Antigen ofMycobacterium tuberculosis

AbstractPolypeptide Ags present in the culture filtrate of Mycobacterium tuberculosis were purified and evaluated for their ability to stimulate PBMC from purified protein derivative (PPD)-positive healthy donors. One such Ag, which elicited strong proliferation and IFN-γ production, was further characterized. The N-terminal amino acid sequence of this polypeptide was determined and used to design oligonucleotides for screening a recombinant M. tuberculosis genomic DNA library. The gene (Mtb 8.4) corresponding to the identified polypeptide was cloned, sequenced, and expressed in Escherichia coli. The predicted m.w. of the recombinant protein without its signal peptide was 8.4 kDa. By Southern analysis, the DNA encoding this mycobacterial protein was found in the M. tuberculosis substrains H37Rv, H37Ra, Erdman, and “C” strain, as well as in certain other mycobacterial species, including Mycobacterium avium and Mycobacterium bovis BCG (bacillus Calmette-Guérin, Pasteur). The Mtb 8.4 gene appears to be absent from the environmental mycobacterial species examined thus far, including Mycobacterium smegmatis, Mycobacterium gordonae, Mycobacterium chelonae, Mycobacterium fortuitum, and Mycobacterium scrofulaceum. Recombinant Mtb 8.4 Ag induced significant proliferation as well as production of IFN-γ, IL-10, and TNF-α, but not IL-5, from human PBMC isolated from PPD-positive healthy donors. Mtb 8.4 did not stimulate PBMC from PPD-negative donors. Furthermore, immunogenicity studies in mice indicate that Mtb 8.4 elicits a Th1 cytokine profile, which is considered important for protective immunity to tuberculosis. Collectively, these results demonstrate that Mtb 8.4 is an immunodominant T cell Ag of M. tuberculosis.