Hepatitis C virus soluble E2 in combination with QuilA and CpG ODN induces neutralizing antibodies in mice

Several studies have emphasized the importance of an early, highly neutralizing antibody response in the clearance of Hepatitis C virus (HCV) infection. The envelope glycoprotein E2 is a major target for HCV neutralizing antibodies. Here, we compared antibody responses in mice immunized with native soluble E2 (sE2) from the H77 1a isolate coupled with different adjuvants or combinations of adjuvants. Adjuvanting sE2 with Freund's, monophosphoryl lipid A (MPL), cytosine phosphorothioate guanine oligodeoxynucleotide (CpG ODN), or alpha-galactosylceramide (αGalCer) derivatives elicited only moderate antibody responses. In contrast, immunizations with sE2 and QuilA elicited exceptionally high anti-E2 antibody titers. Sera from these mice effectively neutralized HCV pseudoparticles (HCVpp) 1a entry. Moreover, the combination of QuilA and CpG ODN further enhanced neutralizing antibody titers wherein cross-neutralization of HCVpp 4 was observed. We conclude that the combination of QuilA and CpG ODN is a promising adjuvant combination that should be further explored for the development of an HCV subunit vaccine. Our work also emphasizes that the ideal combination of adjuvant and immunogen has to be determined empirically.     

Murine neutralizing antibody response and toxicity to synthetic peptides derived from E1 and E2 proteins of hepatitis C virus

Introduction

The highest estimated prevalence of HCV infection has been reported in Egypt, nearly 12% mostly type 4. Currently, a commercial vaccine to protect this high risk population as well as global HCV infected patients is not available.

Objectives

In the present study, we aim at: (1) examining the viral binding capacities of purified monospecific polyclonal murine antibodies raised against genetically conserved viral protein sequences, i.e. synthetic peptides derived from those sequences located within envelope proteins and (2) assessment of immunogenic properties and safety parameters of those peptides individually and in a vaccine format in mice.

Methods

Purified IgG Abs from immunized mice were used in immunocapture RT-PCR experiments to test viral neutralization by Abs raised against each of 4 peptides termed p35 (E1), p36 (E2), p37 (E2) and p38 (E2). Swiss mice were immunized with each of the 3 peptides (p35, p37 and p38) which generated neutralizing antibodies in immunocapture experiments. Antibody responses to corresponding peptides were determined using different routes of administration, different adjuvants, different doses and at different time points post-injection. To explore the dose range for future pharmacological studies, three doses namely 50 ng, 10 μg and 50 μg/25 gm mouse body weight were tested for biochemical and histopathological changes in several organs.

Results

Murine Abs against p35, p37 and p38 but not p36 showed HCV neutralization in immunocapture experiments. Subcutaneous injection of peptides elicited higher responses than i.m. and i.p. Immunization with Multiple Antigenic Peptide (MAP) form or coupled to Al PO4 elicited the highest Ab responses. Peptide doses of 50 ng/25 gm body weight or less were effective and safe, however dose assessment still requires further study. Histopathological changes were observed in animals that received doses ∼1000 times higher than the potential therapeutic dose.

Conclusion

Exploration of humoral immunogenicity, neutralization capacity and safety suggested that the peptides presented herein are candidate vaccine components for further preclinical assessment.     

Therapeutic efficacy of oral immunization with attenuated Salmonella typhimurium expressing Helicobacter pylori CagA, VacA and UreB fusion proteins in mice model

Therapeutic vaccination is a desirable alternative for controlling Helicobacter pylori (H. pylori) infection. In the present study, attenuated Salmonella vector vaccines were constructed that expressed fusion proteins complexed with H. pylori CagA, VacA and UreB in different arrangements, and their therapeutic efficacy was evaluated in H. pylori-infected mice. Oral therapeutic immunization with attenuated Salmonella, which expressed the fused protein CVU, significantly decreased H. pylori colonization in the stomach; protection was related to specific CD4⁺ T cell Th1 type responses and serum IgG and mucosal sIgA antibody responses. These findings suggested that therapeutic efficacy was related to the arrangement of the fusion protein. It is possible that arrangement decides the expression of recombinant antigen in mice, and the latter results in different therapeutic efficacy. The attenuated Salmonella vector vaccine, which expressed the fused protein arrangement CVU, is superior to others, and could be a candidate vaccine against H. pylori.     

Hepatitis B virus core particles displaying Mycobacterium tuberculosis antigen ESAT-6 enhance ESAT-6-specific immune responses

Early secreted antigenic target-6 (ESAT-6), an important Mycobacterium tuberculosis T-cell antigen, is an attractive candidate antigen for tuberculosis subunit vaccine development. Because ESAT-6 has a low inherent immunogenicity, we used Hepatitis B virus core (HBc) protein as an immune carrier to enhance ESAT-6 immunogenicity. The ESAT-6 gene was inserted into the major immunodominant region of the HBc molecule by fusion PCR. The recombinant protein, HBc-ESAT-6 (HE6), was expressed in Escherichia coli, and electron microscopy confirmed the formation of virus-like particles. The immunogenicity of the chimeric particles was assessed in mice. Serological assays and in vitro Th1-biased cytokine assays found that immunization with HE6 particles elicited significantly higher ESAT-6-specific antibodies and CD4⁺/CD8⁺ T cell responses in mice compared to immunization with recombinant ESAT-6 protein. These data demonstrate the feasibility of HBc particles serving as an efficient immune carrier for ESAT-6 and suggest that HE6 has potential for use in a tuberculosis subunit vaccine.     

Immunization of Zika virus envelope protein domain III induces specific and neutralizing immune responses against Zika virus

In this study, we described the generation and immunogenicity of the Zika Virus (ZIKV) envelope protein (E) domain III (DIII) as a protein subunit vaccine candidate. ZIKV EDIII (zEDIII) was rapidly produced in E. coli in inclusion bodies. ZIKV EDIII was solubilized, refolded and purified to >95% homogeneity with a one-step Ni²⁺ affinity chromatography process. Further analysis revealed that zEDIII was refolded properly and demonstrated specific binding to an anti-zEDIII monoclonal antibody that recognizes a zEDIII conformational epitope. Subcutaneous immunization of mice with 25 and 50 μg of zEDIII was performed over a period of 11 weeks. zEDIII evoked ZIKV-specific and neutralizing antibody response with titers that exceed the threshold that correlates with protective immunity against ZIKV. The antigen-specific IgG isotypes were predominantly IgG1 and splenocyte cultures from immunized mice secreted IFN-gamma, IL-4 and IL-6. Notably, zEDIII-elicited antibodies did not enhance the infection of dengue virus in Fc gamma receptor (FcγR)-expressing cells. This study provided a proof of principle for the further development of recombinant protein-based subunit vaccines against ZIKV.     

Long-lasting humoral and cellular immune responses elicited by immunization with recombinant chimeras of the Plasmodium vivax circumsporozoite protein

The circumsporozoite protein (CSP), the most abundant surface antigen of sporozoites, has been extensively studied in different expression platforms as a vaccine candidate. Clinical trials have shown the necessity of broad and highly avid humoral immune responses together with high numbers of CSP-specific T_CD4+ and T_CD8+ cells, especially those producing IFN-γ, to induce protection. To this aim, we designed two distinct recombinant immunogens based on previously-described antigenic fragments of Plasmodium vivax CSP (PvCSP) to be used as vaccine candidates. The first one is a virus-like particle (VLP) comprising the repeat region of PvCSP (B and T_CD4+ epitopes) within the loop of the hepatitis B virus core antigen (HBcAgPvCSP). The second one is a PvCSP multi-epitope polypeptide, rPvCSP-ME, designed based on antigenic regions of PvCSP recognized by lymphocytes of individuals from endemic areas.     

Evaluation of humoral and cellular immune responses to BP26 and OMP31 epitopes in the attenuated Brucella melitensis vaccinated sheep

In recent years, the number of cases of human brucellosis has been increasing by approximately 10% per year in China. Most cases were caused by Brucella melitensis through contacts with infected sheep, goats or their products. An attenuated B. melitensis vaccine M5-90 is currently used to vaccinate both animals in China. This vaccine has not been investigated for critical parameters such as immune response and its association with protective efficacy. In this study, humoral and cellular immune response to the periplasmic protein BP26 and the outer membrane protein OMP31 were evaluated in M5-90 vaccinated Chinese merino and Kazak sheep. Antibodies to BP26 or OMP31 were detected at low levels, and specific IFN-γ response was quantified. Strongly reactive peptides derived from BP26 and OMP31 identified five T-cell epitopes (BP26-6, -8, -11, -12 and OMP31-23) common to both sheep species, five species-specific epitopes (BP26-10, -18, -21 and -22 and OMP31-12) and four animal-specific epitopes (BP26-15, -23, OMP31-6 and -21), which stimulated specific IFN-γ response in vaccinated sheep. Among those T-cell epitopes, reactivity to BP26-18 and -21 epitopes was significantly associated with MHC-I B allele (P = 0.024). However, a specific T-cell response induced by the M5-90 vaccine was relatively week and did not sustain long enough, which might be suppressed by rapid activation of T-regulatory (Treg) cells following vaccination. These findings provide an insight in designing a safer and more effective vaccine for use in animals and in humans.     

Intraperitoneal immunization of recombinant HSP70 (DnaK) of Salmonella Typhi induces a predominant Th2 response and protective immunity in mice against lethal Salmonella infection

Heat shock proteins serve as important antigens in defense against infectious diseases. Members of HSP70 family, particularly microbial HSP70s have acquired special significance in immunity. In the present study, we evaluated the immunogenicity and protective efficacy of HSP70 of Salmonella enterica serovar Typhi against lethal infection by Salmonella in mice with or without adjuvants. The HSP70 gene was cloned and expressed in Escherichia coli BL21 and purified by affinity chromatography. Immunization of mice with HSP70 either alone or in combination with complete Freund's adjuvant (CFA) resulted in a significant increase in antibody titers as compared to control. Antibody isotyping revealed that HSP70 immunization induces both IgG1 and IgG2a antibodies to a significant extent but a higher IgG1/IgG2a ratio indicates a predominant Th2 response. There was a significant increase in lymphocyte proliferation, and levels of both Th2 and Th1 cytokines in cells isolated from immunized mice as compared to control. Immunization of mice with recombinant HSP70 either alone or in combination with CFA conferred 70-90% protection against lethal infections by Salmonella Typhi Ty2 or Salmonella Typhimurium. However, passive immunization with anti-HSP70 sera induced only partial protection in the immunized mice.     

Whole recombinant Hansenula polymorpha expressing hepatitis B virus surface antigen (yeast-HBsAg) induces potent HBsAg-specific Th1 and Th2 immune responses

Recent studies have suggested that yeast cell wall components possess adjuvant activities. In the present study, heat-killed whole recombinant Hansenula polymorpha yeast expressing hepatitis B surface antigen (yeast-HBsAg) was generated, and the immune responses elicited by yeast-HBsAg were investigated in mice. The studies showed that yeast-HBsAg as well as yeast greatly promotes the accumulation of immune cells in mouse spleen and contributes to the maturation of dendritic cells (DCs). Yeast-HBsAg not only induces significantly higher antibody responses (including IgG, IgG1 and IgG2a), but also increases the IgG2a/IgG1 ratio, while alum combined with HBsAg (HBsAg + alum) only enhances antibody responses, but not the IgG2a/IgG1 ratio compared to HBsAg alone. Analysis of HBsAg-specific cytokines revealed that yeast-HBsAg is associated with production of both IFN-γ and IL-4, but neither IFN-γ nor IL-4 was detected in the HBsAg + alum-immunized group. Moreover, yeast-HBsAg induces potent HBsAg-specific lymphocyte proliferation and Cytotoxic T lymphocyte (CTL) responses. In conclusion, yeast-HBsAg enhances both HBsAg-specific Th1 and Th2 immune responses, while alum only enhances Th2 immune responses, suggesting that yeast-HBsAg may be an ideal candidate for an effective vaccine for the control of chronic hepatitis B virus (HBV) infection.     

Platycodin D is a potent adjuvant of specific cellular and humoral immune responses against recombinant hepatitis B antigen

The ideal adjuvants for hepatitis B vaccines should be capable of eliciting both strong humoral and cellular immune responses, especially Th1 cell and cytotoxic T lymphocyte (CTL) responses. However, Alum used as adjuvants in the hepatitis B vaccines currently commercialized offers limitation in inducing cell-mediated response. Therefore, a less hemolytic saponin platycodin D (PD) from the root of Platycodon grandiflorum has been explored for its potential as an alternative adjuvant. In order to compare the adjuvant activity with Alum, antigen-specific cellular and humoral immune responses were evaluated following immunization with a formulation containing hepatitis B surface antigen (HBsAg) adjuvanted with PD and Alum in mice. The Con A-, LPS-, and HBsAg-induced splenocyte proliferation and the serum HBsAg-specific IgG, IgG1, IgG2a, and IgG2b antibody titers in the HBsAg-immunized mice were significantly enhanced by PD (P < 0.05, P < 0.01 or P < 0.001). PD also significantly promoted the production of Th1 (IL-2 and IFN-γ) and Th2 (IL-10) cytokines and up-regulated the mRNA expression of Th1 cytokines (IL-2 and IFN-γ) in splenocytes from the mice immunized with HBsAg (P < 0.001). Besides, PD remarkably increased the killing activities of natural killer (NK) cells and CTLs from splenocytes in the HBsAg-immunized mice (P < 0.001), which may have important implications for vaccination against hepatitis B virus. The results indicated that PD has strong potential to increase both cellular and humoral immune responses and elicit a balanced Th1/Th2 response against HBsAg, and that PD may be the candidates as adjuvants for use in prophylactic and therapeutic hepatitis B vaccine.     

Evaluation of recombinant Chinese avian hepatitis E virus (CaHEV) ORF2 and ORF3 proteins for protection of chickens against CaHEV infection

Avian hepatitis E virus (HEV) is the etiologic agent of big liver and spleen disease in chickens. In 2010, the Chinese avian HEV (CaHEV) strain was isolated from chickens and demonstrated to cause the decreased egg production in layer hens. No avian HEV commercial vaccine has yet been developed to prevent virus infection in China. In this study, recombinant CaHEV truncated ORF2 and complete ORF3 proteins were evaluated separately for immunoprotection of chickens against CaHEV infection. First, truncated ORF2 and complete ORF3 proteins were expressed in Escherichia coli. Next, 48 specific-pathogen-free chickens were randomly divided into three groups. One group was immunized with truncated ORF2 protein, the second group was immunized with recombinant ORF3 protein, while the third group (control) was mock-immunized with PBS. After booster immunization, chickens in all three groups were challenged intravenously with CaHEV infectious stock and assessed for viremia, fecal virus shedding, seroconversion, and gross hepatic lesions. In the ORF2 protein-immunized group, no chickens showed evidence of avian HEV infection. In the ORF3 protein-immunized group, nine chickens exhibited viremia and seven had fecal virus shedding. In the control group, all 16 chickens showed viremia and fecal virus shedding. However, the durations in chickens from the ORF3 protein group (2–4 weeks) were shorter than the ones from the control group (4–8 weeks). Moreover, no gross liver lesions emerged in the ORF2 protein group, while lesions observed in the ORF3 protein group were milder than in controls. Therefore, the ORF2 protein can confer complete immunoprotection against chicken CaHEV infection, while the ORF3 protein only confers partial immunoprotection.     

Vaccination of koalas (Phascolarctos cinereus) with a recombinant chlamydial major outer membrane protein adjuvanted with poly I:C,a host defense peptide and polyphosphazine,elicits strong and long lasting cellular and humoral immune responses

Chlamydial infections are wide spread in koalas across their range and a solution to this debilitating disease has been sought for over a decade. Antibiotics are the currently accepted therapeutic measure, but are not an effective treatment due to the asymptomatic nature of some infections and a low efficacy rate. Thus, a vaccine would be an ideal way to address this infectious disease threat in the wild. Previous vaccine trials have used a three-dose regimen; however this is very difficult to apply in the field as it would require multiple capture events, which are stressful and invasive processes for the koala. In addition, it requires skilled koala handlers and a significant monetary investment. To overcome these challenges, in this study we utilized a polyphosphazine based poly I:C and a host defense peptide adjuvant combined with recombinant chlamydial major outer membrane protein (rMOMP) antigen to induce long lasting (54 weeks) cellular and humoral immunity in female koalas with a novel single immunizing dose. Immunized koalas produced a strong IgG response in plasma, as well as at mucosal sites. Moreover, they showed high levels of C. pecorum specific neutralizing antibodies in the plasma as well as vaginal and conjunctival secretions. Lastly, Chlamydia-specific lymphocyte proliferation responses were produced against both whole chlamydial elementary bodies and rMOMP protein, over the 12-month period. The results of this study suggest that a single dose rMOMP vaccine incorporating a poly I:C, host defense peptide and polyphosphazine adjuvant is able to stimulate both arms of the immune system in koalas, thereby providing an alternative to antibiotic treatment and/or a three-dose vaccine regime.     

Recombinant Ag85B vaccine by taking advantage of characteristics of human parainfluenza type 2 virus vector showed Mycobacteria-specific immune responses by intranasal immunization

Viral vectors are promising vaccine candidates for eliciting suitable Ag-specific immune response. Since Mycobacterium tuberculosis (Mtb) normally enters hosts via the mucosal surface of the lung, the best defense against Mtb is mucosal vaccines that are capable of inducing both systemic and mucosal immunity. Although Mycobacterium bovis bacille Calmette-Guérin is the only licensed tuberculosis (TB) vaccine, its efficacy against adult pulmonary forms of TB is variable. In this study, we assessed the effectiveness of a novel mucosal TB vaccine using recombinant human parainfluenza type 2 virus (rhPIV2) as a vaccine vector in BALB/c mice. Replication-incompetent rhPIV2 (M gene-eliminated) expressing Ag85B (rhPIV2–Ag85B) was constructed by reverse genetics technology. Intranasal administration of rhPIV2–Ag85B induced Mtb-specific immune responses, and the vaccinated mice showed a substantial reduction in the number of CFU of Mtb in lungs and spleens. Unlike other viral vaccine vectors, the immune responses against Ag85B induced by rhPIV2–Ag85B immunization had an advantage over that against the viral vector. In addition, it was revealed that rhPIV2–Ag85B in itself has an adjuvant activity through the retinoic acid-inducible gene I receptor. These findings provide further evidence for the possibility of rhPIV2–Ag85B as a novel TB vaccine.