Optimisation of prime-boost immunization in mice using novel protein-based and recombinant vaccinia (tiantan)-based HBV vaccine

Background

A therapeutic vaccine for chronic hepatitis B virus (HBV) infection that enhances virus-specific cellular immune responses is urgently needed. The “prime–boost” regimen is a widely used vaccine strategy against many persistence infections. However, few reports have addressed this strategy applying for HBV therapeutic vaccine development.

Methodology/Principal Findings

To develop an effective HBV therapeutic vaccine, we constructed a recombinant vaccinia virus (Tiantan) containing the S+PreS1 fusion antigen (RVJSS1) combined with the HBV particle-like subunit vaccine HBVSS1 to explore the most effective prime–boost regimen against HBV. The immune responses to different prime–boost regimens were assessed in C57BL/C mice by ELISA, ELISpot assay and Intracellular cytokine staining analysis. Among the combinations tested, an HBV protein particle vaccine priming and recombinant vaccinia virus boosting strategy accelerated specific seroconversion and produced high antibody (anti-PreS1, anti-S antibody) titres as well as the strongest multi-antigen (PreS1, and S)-specific cellular immune response. HBSS1 protein prime/RVJSS1 boost immunization was also generated more significant level of both CD4+ and CD8+ T cell responses for Th1 cytokines (TNF-α and IFN-γ).

Conclusions

The HBSS1 protein-vaccine prime plus RVJSS1 vector boost elicits specific antibody as well as CD4 and CD8 cells secreting Th1-like cytokines, and these immune responses may be important parameters for the future HBV therapeutic vaccines.     

DNA immunization with fusion of CTLA-4 to hepatitis B virus (HBV) core protein enhanced Th2 type responses and cleared HBV with an accelerated kinetic

Background

Typically, DNA immunization via the intramuscular route induces specific, Th1-dominant immune responses. However, plasmids expressing viral proteins fused to cytotoxic T lymphocyte antigen 4 (CTLA-4) primed Th2-biased responses and were able to induced effective protection against viral challenge in the woodchuck model. Thus, we addressed the question in the mouse model how the Th1/Th2 bias of primed immune responses by a DNA vaccine influences hepatitis B virus (HBV) clearance.

Principal Findings

Plasmids expressing HBV core protein (HBcAg) or HBV e antigen and HBcAg fused to the extracellular domain of CTLA-4 (pCTLA-4-HBc), CD27, and full length CD40L were constructed. Immunizations of these DNA plasmids induced HBcAg-specific antibody and cytotoxic T-cell responses in mice, but with different characteristics regarding the titers and subtypes of specific antibodies and intensity of T-cell responses. The plasmid pHBc expressing HBcAg induced an IgG2a-dominant response while immunizations of pCTLA-4-HBc induced a balanced IgG1/IgG2a response. To assess the protective values of the immune responses of different characteristics, mice were pre-immunized with pCTLA-4-HBc and pHBc, and challenged by hydrodynamic injection (HI) of pAAV/HBV1.2. HBV surface antigen (HBsAg) and DNA in peripheral blood and HBcAg in liver tissue were cleared with significantly accelerated kinetics in both groups. The clearance of HBsAg was completed within 16 days in immunized mice while more than 50% of the control mice are still positive for HBsAg on day 22. Stronger HBcAg-specific T-cell responses were primed by pHBc correlating with a more rapid decline of HBcAg expression in liver tissue, while anti-HBs antibody response developed rapidly in the mice immunized with pCTLA-4-HBc, indicating that the Th1/Th2 bias of vaccine-primed immune responses influences the mode of viral clearance.

Conclusion

Viral clearance could be efficiently achieved by Th1/Th2-balanced immune response, with a small but significant shift in T-cell and B-cell immune responses.     

Resiquimod and polyinosinic–polycytidylic acid formulation with aluminum hydroxide as an adjuvant for foot-and-mouth disease vaccine

Background

Toll-like receptor (TLR) agonists reportedly have potent antiviral and antitumor activities and may be a new kind of adjuvant for enhancing immune efficacy. Resiquimod (R848) is an imidazoquinoline compound with potent antiviral activity and functions through the TLR7/TLR8 MyD88-dependent signaling pathway. Polyinosinic-polycytidylic acid [poly(I:C)] is a synthetic analog of double-stranded RNA that induces the production of pro-inflammatory cytokines by the activation of NF-κB through TLR3. This study investigated the potential of R848 and poly(I:C) as an adjuvant 146S foot-and-mouth disease virus (FMDV) vaccine formulated with aluminum hydroxide (Al(OH)₃).

Results

Antibody titers to FMDV and CD8⁺ T cells were markedly enhanced in mice immunized to 146S FMDV?+?Al(OH)₃?+?R848?+?poly(I:C) compared with mice immunized to FMDV?+?ISA206. IFN-γ secretion substantially increased compared with IL-4 secretion by splenic T cells stimulated with FMDV antigens in vitro, suggesting that R848, poly(I:C), and with Al(OH)₃ together biased the immune response toward a Th1-type direction.

Conclusions

These results indicated that the R848 and poly(I:C) together with Al(OH)₃ enhanced humoral and cellular immune responses to immunization with 146S FMDV antigens. Thus, this new vaccine formulation can be used for FMDV prevention.     

Modified Vaccinia Virus Ankara Exerts Potent Immune Modulatory Activities in a Murine Model

Background

Modified vaccinia virus Ankara (MVA), a highly attenuated strain of vaccinia virus, has been used as vaccine delivery vector in preclinical and clinical studies against infectious diseases and malignancies. Here, we investigated whether an MVA which does not encode any antigen (Ag) could be exploited as adjuvant per se.

Methodology/Principal Findings

We showed that dendritic cells infected in vitro with non-recombinant (nr) MVA expressed maturation and activation markers and were able to efficiently present exogenously pulsed Ag to T cells. In contrast to the dominant T helper (Th) 1 biased responses elicited against Ags produced by recombinant MVA vectors, the use of nrMVA as adjuvant for the co-administered soluble Ags resulted in a long lasting mixed Th1/Th2 responses.

Conclusions/Significance

These findings open new ways to potentiate and modulate the immune responses to vaccine Ags depending on whether they are co-administered with MVA or encoded by recombinant viruses.     

Chronic Schistosoma japonicum Infection Reduces Immune Response to Vaccine against Hepatitis B in Mice

Background

Hepatitis B and schistosomiasis are most prevalent in Africa and Asia, and co-infections of both are frequent in these areas. The immunomodulation reported to be induced by schistosome infections might restrict immune control of hepatitis B virus (HBV) leading to more severe viral infection. Vaccination is the most effective measure to control and prevent HBV infection, but there is evidence for a reduced immune response to the vaccine in patients with chronic schistosomiasis japonica.

Methodology/Principal Findings

In this paper, we demonstrate in a mouse model that a chronic Schistosoma japonicum infection can inhibit the immune response to hepatitis B vaccine (HBV vaccine) and lead to lower production of anti-HBs antibodies, interferon-γ (IFN-γ) and interleukin-2 (IL-2). After deworming with Praziquantel (PZQ), the level of anti-HBs antibodies gradually increased and the Th2-biased profile slowly tapered. At 16 weeks after deworming, the levels of anti-HBs antibodies and Th1/Th2 cytokines returned to the normal levels.

Conclusions/Significance

The results suggest that the preexisting Th2-dominated immune profile in the host infected with the parasite may down–regulate levels of anti-HBs antibodies and Th1 cytokines. To improve the efficacy of HBV vaccination in schistosome infected humans it may be valuable to treat them with praziquantel (PZQ) some time prior to HBV vaccination.     

Immunization of Aged Pigs with Attenuated Pseudorabies Virus Vaccine Combined with CpG Oligodeoxynucleotide Restores Defective Th1 Immune Responses

Background and Aims

Attempts to immunize aged subjects often result in the failure to elicit a protective immune response. Murine model studies have shown that oligonucleotides containing CpG motifs (CpG ODN) can stimulate immune system in aged mice as effectively as in young mice. Since many physiological and pathophysiological data of pigs can be transferred to humans, research in pigs is important to confirm murine data. Here we investigated whether immunization of aged pig model with attenuated pseudorabies virus vaccine (PRV vaccine) formulated with CpG ODN could promote a successful development of immune responses that were comparable to those induced in young pigs in a similar manner.

Methodology

Young and aged pigs were immunized IM with PRV vaccine alone, or in combination with CpG ODN respectively. At days 3, 7, 14 post immunization sera were assayed by ELISA for IgG titres, at day 7 for IgG1 and IgG2 subtypes titres. All blood samples collected in evacuated test tubes with K-EDTA at day 7 were analyzed for flow cytometer assay. Blood samples at day 7 collected in evacuated test tubes with heparin were analysed for antigen-specific cytokines production and peripheral blood mononuclear cells (PBMCs) proliferative responses.

Results

CpG ODN could enhance Th1 responses (PRV-specific IgG2/IgG1 ratio, proliferative responses, Th1 cytokines production) when used as an adjuvant for the vaccination of aged pigs, which were correlated with enhanced CD4+ T cells percentage, decreased CD4+CD8+CD45RO+ T cells percentage and improved PRV-specific CD4+ T cells activation.

Conclusions

Our results demonstrate a utility for CpG ODN, as a safe vaccine adjuvant for promoting effective systemic immune responses in aged pig model. This agent could have important clinical uses in overcoming some of age-associated depressions in immune function that occur in response to vaccination.     

Immunization with SARS coronavirus vaccines leads to pulmonary immunopathology on challenge with the SARS virus

Background

Severe acute respiratory syndrome (SARS) emerged in China in 2002 and spread to other countries before brought under control. Because of a concern for reemergence or a deliberate release of the SARS coronavirus, vaccine development was initiated. Evaluations of an inactivated whole virus vaccine in ferrets and nonhuman primates and a virus-like-particle vaccine in mice induced protection against infection but challenged animals exhibited an immunopathologic-type lung disease.

Design

Four candidate vaccines for humans with or without alum adjuvant were evaluated in a mouse model of SARS, a VLP vaccine, the vaccine given to ferrets and NHP, another whole virus vaccine and an rDNA-produced S protein. Balb/c or C57BL/6 mice were vaccinated IM on day 0 and 28 and sacrificed for serum antibody measurements or challenged with live virus on day 56. On day 58, challenged mice were sacrificed and lungs obtained for virus and histopathology.

Results

All vaccines induced serum neutralizing antibody with increasing dosages and/or alum significantly increasing responses. Significant reductions of SARS-CoV two days after challenge was seen for all vaccines and prior live SARS-CoV. All mice exhibited histopathologic changes in lungs two days after challenge including all animals vaccinated (Balb/C and C57BL/6) or given live virus, influenza vaccine, or PBS suggesting infection occurred in all. Histopathology seen in animals given one of the SARS-CoV vaccines was uniformly a Th2-type immunopathology with prominent eosinophil infiltration, confirmed with special eosinophil stains. The pathologic changes seen in all control groups lacked the eosinophil prominence.

Conclusions

These SARS-CoV vaccines all induced antibody and protection against infection with SARS-CoV. However, challenge of mice given any of the vaccines led to occurrence of Th2-type immunopathology suggesting hypersensitivity to SARS-CoV components was induced. Caution in proceeding to application of a SARS-CoV vaccine in humans is indicated.     

Transient Facial Nerve Paralysis (Bell's Palsy) following Intranasal Delivery of a Genetically Detoxified Mutant of Escherichia coli Heat Labile Toxin

Background

An association was previously established between facial nerve paralysis (Bell''s palsy) and intranasal administration of an inactivated influenza virosome vaccine containing an enzymatically active Escherichia coli Heat Labile Toxin (LT) adjuvant. The individual component(s) responsible for paralysis were not identified, and the vaccine was withdrawn.

Methodology/Principal Findings

Subjects participating in two contemporaneous non-randomized Phase 1 clinical trials of nasal subunit vaccines against Human Immunodeficiency Virus and tuberculosis, both of which employed an enzymatically inactive non-toxic mutant LT adjuvant (LTK63), underwent active follow-up for adverse events using diary-cards and clinical examination. Two healthy subjects experienced transient peripheral facial nerve palsies 44 and 60 days after passive nasal instillation of LTK63, possibly a result of retrograde axonal transport after neuronal ganglioside binding or an inflammatory immune response, but without exaggerated immune responses to LTK63.

Conclusions/Significance

While the unique anatomical predisposition of the facial nerve to compression suggests nasal delivery of neuronal-binding LT–derived adjuvants is inadvisable, their continued investigation as topical or mucosal adjuvants and antigens appears warranted on the basis of longstanding safety via oral, percutaneous, and other mucosal routes.     

The Ocular Conjunctiva as a Mucosal Immunization Route: A Profile of the Immune Response to the Model Antigen Tetanus Toxoid

Background

In a quest for a needle-free vaccine administration strategy, we evaluated the ocular conjunctiva as an alternative mucosal immunization route by profiling and comparing the local and systemic immune responses to the subcutaneous or conjunctival administration of tetanus toxoid (TTd), a model antigen.

Materials and methods

BALB/c and C57BL/6 mice were immunized either subcutaneously with TTd alone or via the conjunctiva with TTd alone, TTd mixed with 2% glycerol or TTd with merthiolate-inactivated whole-cell B. pertussis (wBP) as adjuvants. Mice were immunized on days 0, 7 and 14 via both routes, and an evaluation of the local and systemic immune responses was performed two weeks after the last immunization. Four weeks after the last immunization, the mice were challenged with a lethal dose (2 × LD₅₀) of tetanus toxin.

Results

The conjunctival application of TTd in BALB/c mice induced TTd-specific secretory IgA production and skewed the TTd-specific immune response toward a Th1/Th17 profile, as determined by the stimulation of IFNγ and IL-17A secretion and/or the concurrent pronounced reduction of IL-4 secretion, irrespective of the adjuvant. In conjunctivaly immunized C57BL/6 mice, only TTd administered with wBP promoted the establishment of a mixed Th1/Th17 TTd-specific immune response, whereas TTd alone or TTd in conjunction with glycerol initiated a dominant Th1 response against TTd. Immunization via the conjunctiva with TTd plus wBP adjuvant resulted in a 33% survival rate of challenged mice compared to a 0% survival rate in non-immunized animals (p<0.05).

Conclusion

Conjunctival immunization with TTd alone or with various adjuvants induced TTd-specific local and systemic immune responses, predominantly of the Th1 type. The strongest immune responses developed in mice that received TTd together with wBP, which implies that this alternative route might tailor the immune response to fight intracellular bacteria or viruses more effectively.     

Influence of adjuvants in inducing immune responses to different epitopes included in a multiepitope,multivalent, multistage Plasmodium falciparum candidate vaccine (FALVAC-1) in outbred mice

FALVAC-1, a vaccine against Plasmodium falciparum was developed by joining 21 epitopes from P. falciparum vaccine antigens and an universal T helper epitope from tetanus toxoid. Since adjuvants influence different aspects of immune responses, in this study we investigated the effect of four adjuvants aluminum hydroxide (alum), nonionic copolymer adjuvant P1005 (water-in-oil emulsion), CpG oligodeoxynucleotides (ODN), and QS-21 in eliciting immune responses in outbred mice. QS-21 and copolymer adjuvants were the best formulations in inducing higher and long-lasting antibody titers to the whole vaccine compared to alum and CpG. QS-21 was the only adjuvant to elicit predominantly IgG2a response and antibodies reactive with all epitopes incorporated in the vaccine construct. Vaccine elicited antibodies recognized sporozoites and asexual blood-stage parasites. FALVAC-1 immunized mice induced lymphoproliferative and IFN-gamma response to the vaccine. QS-21 and CpG adjuvants were able to elicit T proliferative responses to 20 of the 22 epitopes in the vaccine. In conclusion, this study demonstrated that with suitable adjuvant such as QS-21, it is possible to elicit immune responses to most of the epitopes included in the FALVAC-1 vaccine.     

CpG对乙型肝炎基因重组(CHO细胞)疫苗免疫效果的影响

为了研究CpG-寡脱氧核苷酸(CpG-OPN)作为佐剂对乙型肝炎基因重组(CHO细胞)疫苗(简称乙肝疫苗)免疫效果的影响,以乙肝疫苗加Al(OH)3、疫苗加CpG和疫苗加Al(OH)3与CpG3三种配伍方式,通过腹腔、皮下或肌内3种不同途径免疫Balb/c小鼠,观察不同免疫途径和不同配伍的免疫效果.同时又将疫苗与CpG混合后在4℃存放6个月再免疫小鼠,观察CpG的稳定性.结果表明:①3种免疫途径中以肌内注射效果最好,这在使用CpG的实验组尤为明显,在该组肌内免疫的ED50比腹腔的低了10倍,而诱发的抗体滴度提高了3倍;②疫苗与CpG、Al(OH)3联合使用的免疫效果最好,在肌内免疫时联合使用的免疫效果比疫苗+Al(OH)3提高4倍,比疫苗+CpG提高7倍;③疫苗+Al(OH)3免疫时,表现为IgG1抗体亚型占优势,而再加入CpG后则IgG1和IgG2a均升高,以IgG2a最显著;④疫苗与CpG混合后4℃保存半年,不影响其活性.     

Cationic liposomes formulated with synthetic mycobacterial cordfactor (CAF01): a versatile adjuvant for vaccines with different immunological requirements

Background

It is now emerging that for vaccines against a range of diseases including influenza, malaria and HIV, the induction of a humoral response is insufficient and a substantial complementary cell-mediated immune response is necessary for adequate protection. Furthermore, for some diseases such as tuberculosis, a cellular response seems to be the sole effector mechanism required for protection. The development of new adjuvants capable of inducing highly complex immune responses with strong antigen-specific T-cell responses in addition to antibodies is therefore urgently needed.

Methods and Findings

Herein, we describe a cationic adjuvant formulation (CAF01) consisting of DDA as a delivery vehicle and synthetic mycobacterial cordfactor as immunomodulator. CAF01 primes strong and complex immune responses and using ovalbumin as a model vaccine antigen in mice, antigen specific cell-mediated- and humoral responses were obtained at a level clearly above a range of currently used adjuvants (Aluminium, monophosphoryl lipid A, CFA/IFA, Montanide). This response occurs through Toll-like receptor 2, 3, 4 and 7-independent pathways whereas the response is partly reduced in MyD88-deficient mice. In three animal models of diseases with markedly different immunological requirement; Mycobacterium tuberculosis (cell-mediated), Chlamydia trachomatis (cell-mediated/humoral) and malaria (humoral) immunization with CAF01-based vaccines elicited significant protective immunity against challenge.

Conclusion

CAF01 is potentially a suitable adjuvant for a wide range of diseases including targets requiring both CMI and humoral immune responses for protection.     

Intranasal immunization with recombinant HA and mast cell activator C48/80 elicits protective immunity against 2009 pandemic H1N1 influenza in mice

Background

Pandemic influenza represents a major threat to global health. Vaccination is the most economic and effective strategy to control influenza pandemic. Conventional vaccine approach, despite being effective, has a number of major deficiencies including limited range of protection, total dependence on embryonated eggs for production, and time consuming for vaccine production. There is an urgent need to develop novel vaccine strategies to overcome these deficiencies.

Methodology/Principal Findings

The major objective of this work was to develop a novel vaccine strategy combining recombinant haemagglutinin (HA) protein and a master cell (MC) activator C48/80 for intranasal immunization. We demonstrated in BALB/c mice that MC activator C48/80 had strong adjuvant activity when co-administered with recombinant HA protein intranasally. Vaccination with C48/80 significantly increased the serum IgG and mucosal surface IgA antibody responses against HA protein. Such increases correlated with stronger and durable neutralizing antibody activities, offering protection to vaccinated animals from disease progression after challenge with lethal dose of A/California/04/2009 live virus. Furthermore, protected animals demonstrated significant reduction in lung virus titers, minimal structural alteration in lung tissues as well as higher and balanced production of Th1 and Th2 cytokines in the stimulated splenocytes when compared to those without C48/80.

Conclusions/Significance

The present study demonstrates that the novel vaccine approach of combining recombinant HA and mucosal adjuvant C48/80 is safe and effective in eliciting protective immunity in mice. Future studies on the mechanism of action of C48/80 and potential combination with other vaccine strategies such as prime and boost approach may help to induce even more potent and broad immune responses against viruses from various clades.     

Immunogenicity of Live Attenuated B. pertussis BPZE1 Producing the Universal Influenza Vaccine Candidate M2e

Background

Intranasal delivery of vaccines directed against respiratory pathogens is an attractive alternative to parenteral administration. However, using this delivery route for inactivated vaccines usually requires the use of potent mucosal adjuvants, and no such adjuvant has yet been approved for human use.

Methodology/Principal Findings

We have developed a live attenuated Bordetella pertussis vaccine, called BPZE1, and show here that it can be used to present the universal influenza virus epitope M2e to the mouse respiratory tract to prime for protective immunity against viral challenge. Three copies of M2e were genetically fused to the N-terminal domain of filamentous hemagglutinin (FHA) and produced in recombinant BPZE1 derivatives in the presence or absence of endogenous full-length FHA. Only in the absence of FHA intranasal administration of the recombinant BPZE1 derivative induced antibody responses to M2e and effectively primed BALB/c mice for protection against influenza virus-induced mortality and reduced the viral load after challenge. Strong M2e-specific antibody responses and protection were observed after a single nasal administration with the recombinant BPZE1 derivative, followed by a single administration of M2e linked to a virus-like particle without adjuvant, whereas priming alone with the vaccine strain did not protect.

Conclusions/Significance

Using recombinant FHA-3M2e-producing BPZE1 derivatives for priming and the universal influenza M2e peptide linked to virus-like particles for boosting may constitute a promising approach for needle-free and adjuvant-free nasal vaccination against influenza.     

Parenteral and mucosal prime-boost immunization strategies in mice with hepatitis B surface antigen and CpG DNA

Synthetic oligodeoxynucleotides (ODN) containing immunostimulatory CpG motifs (CpG ODN) are potent adjuvants to protein antigens administered by parenteral or mucosal routes to BALB/c mice. To date, there have been no studies using combined parenteral/mucosal approaches with CpG DNA as adjuvant. In this study we evaluated different parenteral prime-mucosal boost and mucosal prime-parenteral boost strategies using hepatitis B surface antigen (HBsAg) alone or with different adjuvants: aluminum hydroxide (alum), cholera toxin (CT), CpG ODN. In addition, since CpG ODN has previously been shown to act synergistically with other adjuvants after parenteral or mucosal delivery, we also evaluated adjuvant combinations: alum+CpG ODN and CT+CpG ODN. The effects of adjuvant and administration strategy on systemic and mucosal humoral responses were measured, as well as cell-mediated immune responses (cytotoxic T lymphocyte activity). These results were compared to parenteral only or mucosal only strategies. Our findings demonstrate that parenteral immunization can prime for mucosal responses even when different lymph nodes were being targeted. HBsAg-specific immune responses (IgG in plasma, cytotoxic T lymphocytes) induced by parenteral prime could all be significantly enhanced by mucosal boosting and despite the fact that intramuscular immunization alone could not induce mucosal IgA, it could prime for a subsequent mucosal boost. In addition, the presence of adjuvant at time of boosting could influence the nature of subsequent immune responses (Th1 vs. Th2). Mice primed intranasally could have their systemic immune responses boosted with a parenteral administration and it was also possible to enhance mucosal responses induced by intranasal prime with an intramuscular boost.     

Adult-like anti-mycobacterial T cell and in vivo dendritic cell responses following neonatal immunization with Ag85B-ESAT-6 in the IC31 adjuvant

Background

With the exception of some live vaccines, e.g. BCG, subunit vaccines formulated with “classical” adjuvants do not induce similar responses in neonates as in adults. The usual neonatal profile is characterized by lower levels of TH1-associated biomarkers. This has hampered the development of new neonatal vaccines for diseases that require early protection. Tuberculosis is one of the major targets for neonatal immunization. In this study, we assessed the immunogenicity of a novel candidate vaccine comprising a mycobacterial fusion protein, Ag85B-ESAT-6, in a neonatal murine immunization model.

Methods/Findings

The Ag85B-ESAT-6 fusion protein was formulated either with a classical alum based adjuvant or with the novel IC31® adjuvant. Following neonatal or adult immunization, 3 parameters were studied in vivo: (1) CD4⁺ T cell responses, (2) vaccine targeting/activation of dendritic cells (DC) and (3) protection in a surrogate mycobacterial challenge model. Conversely to Alum, IC31® induced in both age groups strong Th1 and Th17 responses, characterized by multifunctional T cells expressing IL-2 and TNF-α with or without IFN-γ. In the draining lymph nodes, a similarly small number of DC contained the adjuvant and/or the antigen following neonatal or adult immunization. Expression of CD40, CD80, CD86 and IL-12p40 production was focused on the minute adjuvant-bearing DC population. Again, DC targeting/activation was similar in adults and neonates. These DC/T cell responses resulted in an equivalent reduction of bacterial growth following infection with M. bovis BCG, whereas no protection was observed when Alum was used as adjuvant.

Conclusion

Neonatal immunization with the IC31®- adjuvanted Ag85B-ESAT-6 subunit vaccine elicited adult-like multifunctional protective anti-mycobacterial T cell responses through the induction of an adult pattern of in vivo DC activation.     

Characterization of cross protection of Swine-Origin Influenza Virus (S-OIV) H1N1 and reassortant H5N1 influenza vaccine in BALB/c mice given a single-dose vaccination

Background

Influenza virus has antigen drift and antigen shift effect, vaccination with some influenza vaccine might not induce sufficient immunity for host to the threat of other influenza virus strains. S-OIV H1N1 and H5N1 influenza vaccines in single-dose immunization were evaluated in mice for cross protection to the challenge of A/California/7/2009 H1N1 or NIBRG-14 H5N1 virus.

Results

Both H1N1 and H5N1 induced significant homologous IgG, HAI, and microneutralization antibody responses in the mice, while only vaccines plus adjuvant produced significant heterogeneous IgG and HAI antibody responses. Both alum and MPLA adjuvants significantly reduced the S-OIV H1N1 vaccine dose required to elicit protective HAI antibody titers from 0.05 μg to 0.001 μg. Vaccines alone did not protect mice from challenge with heterogeneous influenza virus, while H5N1 vaccine plus alum and MPLA adjuvants did. Mouse body weight loss was also less significant in the presence of adjuvant than in the vaccine without adjuvant. Furthermore, both H1N1 and H5N1 lung viral titers of immunized mice were significantly reduced post challenge with homologous viruses.

Conclusion

Only in the presence of MPLA adjuvant could the H5N1 vaccine significantly reduce mouse lung viral titers post H1N1 virus challenge, and not vice versa. MPLA adjuvant induced cross protection with a single dose vaccination to the challenge of heterogeneous influenza virus in mice. Lung viral titer seemed to be a better indicator compared to IgG, neutralization antibody, and HAI titer to predict survival of mice infected with influenza virus.     

Tailoring Subunit Vaccine Immunity with Adjuvant Combinations and Delivery Routes Using the Middle East Respiratory Coronavirus (MERS-CoV) Receptor-Binding Domain as an Antigen

The development of an effective vaccine is critical for prevention of a Middle East respiratory syndrome coronavirus (MERS-CoV) pandemic. Some studies have indicated the receptor-binding domain (RBD) protein of MERS-CoV spike (S) is a good candidate antigen for a MERS-CoV subunit vaccine. However, highly purified proteins are typically not inherently immunogenic. We hypothesised that humoral and cell-mediated immunity would be improved with a modification of the vaccination regimen. Therefore, the immunogenicity of a novel MERS-CoV RBD-based subunit vaccine was tested in mice using different adjuvant formulations and delivery routes. Different vaccination regimens were compared in BALB/c mice immunized 3 times intramuscularly (i.m.) with a vaccine containing 10 µg of recombinant MERS-CoV RBD in combination with either aluminium hydroxide (alum) alone, alum and polyriboinosinic acid (poly I:C) or alum and cysteine-phosphate-guanine (CpG) oligodeoxynucleotides (ODN). The immune responses of mice vaccinated with RBD, incomplete Freund’s adjuvant (IFA) and CpG ODN by a subcutaneous (s.c.) route were also investigated. We evaluated the induction of RBD-specific humoral immunity (total IgG and neutralizing antibodies) and cellular immunity (ELISpot assay for IFN-γ spot-forming cells and splenocyte cytokine production). Our findings indicated that the combination of alum and CpG ODN optimized the development of RBD-specific humoral and cellular immunity following subunit vaccination. Interestingly, robust RBD-specific antibody and T-cell responses were induced in mice immunized with the rRBD protein in combination with IFA and CpG ODN, but low level of neutralizing antibodies were elicited. Our data suggest that murine immunity following subunit vaccination can be tailored using adjuvant combinations and delivery routes. The vaccination regimen used in this study is promising and could improve the protection offered by the MERS-CoV subunit vaccine by eliciting effective humoral and cellular immune responses.     

A pilot study on developing mucosal vaccine against alveolar echinococcosis (AE) using recombinant tetraspanin 3: Vaccine efficacy and immunology

Background

We have previously evaluated the vaccine efficacies of seven tetraspanins of Echinococcus multilocularis (Em-TSP1–7) against alveolar echinococcosis (AE) by subcutaneous (s.c.) administration with Freund''s adjuvant. Over 85% of liver cyst lesion number reductions (CLNR) were achieved by recombinant Em-TSP1 (rEm-TSP1) and -TSP3 (rEm-TSP3). However, to develop an efficient and safe human vaccine, the efficacy of TSP mucosal vaccines must be thoroughly evaluated.

Methodology/Principal Findings

rEm-TSP1 and -TSP3 along with nontoxic CpG ODN (CpG oligodeoxynucleotides) adjuvant were intranasally (i.n.) immunized to BALB/c mice and their vaccine efficacies were evaluated by counting liver CLNR (experiment I). 37.1% (p<0.05) and 62.1% (p<0.001) of CLNR were achieved by these two proteins, respectively. To study the protection-associated immune responses induced by rEm-TSP3 via different immunization routes (i.n. administration with CpG or s.c. immunization with Freund''s adjuvant), the systemic and mucosal antibody responses were detected by ELISA (experiment II). S.c. and i.n. administration of rEm-TSP3 achieved 81.9% (p<0.001) and 62.8% (p<0.01) CLNR in the liver, respectively. Both the immunization routes evoked strong serum IgG, IgG1 and IgG2α responses; i.n. immunization induced significantly higher IgA responses in nasal cavity and intestine compared with s.c. immunization (p<0.001). Both immunization routes induced extremely strong liver IgA antibody responses (p<0.001). The Th1 and Th2 cell responses were assessed by examining the IgG1/IgG2α ratio at two and three weeks post-immunization. S.c. immunization resulted in a reduction in the IgG1/IgG2α ratio (Th1 tendency), whereas i.n. immunization caused a shift from Th1 to Th2. Moreover, immunohistochemistry showed that Em-TSP1 and -TSP3 were extensively located on the surface of E. multilocularis cysts, protoscoleces and adult worms with additional expression of Em-TSP3 in the inner part of protoscoleces and oncospheres.

Conclusions

Our study indicated that i.n. administration of rEm-TSP3 with CpG is able to induce both systemic and local immune responses and thus provides significant protection against AE.