一种CpG佐剂增强HIV-1DNA疫苗免疫原性的研究

目的以一种CpG寡聚核苷酸为HIV-1DNA疫苗候选佐剂,研究该CpG佐剂增强DNA疫苗免疫原性,体外促进DC细胞成熟等特点。方法在Balb/c小鼠模型上连续3次联合免疫HIV-1DNA疫苗及CpG佐剂,通过IFN-γ、IL-2ELISPOT及ELISA检测HIV特异性细胞免疫反应及体液免疫应答强度;体外制备小鼠骨髓来源的树突状细胞,通过FACS技术、高通量细胞因子检测等方法评价CpG佐剂刺激活化DC的能力。结果 CpG能够增强HIV-1DNA疫苗诱导的特异性细胞免疫反应水平,降低DNA疫苗使用剂量;CpG体外刺激原代小鼠骨髓来源的树突状细胞(BMDC),能显著上调CD40、CD80、CD86等BMDC表面共刺激分子的表达,活化BMDC并分泌各型细胞因子IL-5、IL-12p70,促炎症因子IL-1α、IL-1β、IL-6、IL-10、MIP-2、KC、MIG、Eotaxin、GM-CSF等以发挥佐剂效应。结论综合体内体外实验数据,证实该型CpG能够充分活化BMDC,显著提高HIV-1DNA疫苗免疫原性,降低疫苗使用剂量,可成为HIV-1DNA疫苗临床试验用候选佐剂。     

Synthetic HIV-1 matrix protein p17-based AT20-KLH therapeutic immunization in HIV-1-infected patients receiving antiretroviral treatment: A phase I safety and immunogenicity study

Background

Therapeutic vaccination is a promising novel approach to treat HIV-1 infected people by boosting or redirecting immune system to neutralize critical HIV-1 antigens whose biological effects are relevant in the context of viral pathogenesis. With the aim to induce neutralizing antibodies to the matrix protein p17 we have developed a peptide-based immunogen (AT20-KLH) and evaluated its safety and immunogenicity.

Methodology

Twenty four asymptomatic HAART-treated HIV-1+ patients were enrolled in a phase I clinical study and were randomized to three groups: 2 groups were treated with five IM injection (Arm A: 25 μg/inoculation; Arm B: 100 μg/inoculation) at day (D) D0, D28, D56, D84 and D112; the control group (Arm C) were not injected. Safety was assessed by monitoring local and systemic adverse events (AEs), recorded till D168. Evaluation of immunogenicity was by titering antibodies at D0, D35, D56, D63, D84, D91, D112, D140 and D168 using ELISA.

Results

In all, 105 local and systemic AEs were reported across the three groups. Most were mild and resolved without sequelae. Also the few unsolicited events, deemed unrelated to the study vaccines, caused no problems. No significant changes in the routine laboratory parameters, CD4 T-cell count or HIV-1 viremia were found. At the time of enrollment 23 out of 24 patients had no anti-AT20 antibodies, whereas 11 exhibited anti-p17 antibodies. Irrespective of the presence of preimmunization antibodies, all subjects developed high titers of anti-AT20 antibodies (GM 9775) in response to both AT20-KLH doses. These antibodies were also capable of recognizing AT20 within the p17 framework.

Conclusions

The AT20 peptide-based approach has allowed to redirect HAART-treated patients’ humoral responses toward a previously untargeted hotspot of functional activity. Overall, the tested AT20-KLH doses were safe and well tolerated, supporting further exploration of AT20-KLH as an HIV-1 therapeutic vaccine candidate.     

Type I IFN signalling is required for cationic adjuvant formulation (CAF)01-induced cellular immunity and mucosal priming

Despite being in the midst of a global pandemic of infections caused by the pathogen Chlamydia trachomatis, a vaccine capable of inducing protective immunity remains elusive. Given the C. trachomatis mucosal port of entry, a formulation compatible with mucosal administration and capable of eliciting potent genital tract immunity is highly desirable. While subunit vaccines are considered safer and better tolerated, these are typically poorly immunogenic and require co-formulation with immune-potentiating adjuvants. However, of the adjuvants licensed for use in humans, very few drive robust cellular responses, a pre-requisite for protection against C. trachomatis infection. Recently, the cationic adjuvant formulations (CAF) have been shown to induce robust humoral and cellular immunity in pre-clinical models of chlamydia, malaria and tuberculosis (TB). Here, we demonstrate that CAF01 induces potent immune responses when combined with the major outer membrane protein (MOMP) of C. trachomatis following parenteral immunisation and also as part of a heterologous prime/boost regime. We show that a subcutaneous prime with CAF01-adjuvanted recombinant MOMP licenses antigen-specific immunity at distant mucosal sites which can be activated following oral antigen re-encounter in the absence of concomitant adjuvant stimulation. Finally, we shed light on the mechanism(s) through which CAF01 elicits robust antigen-specific immunity to co-formulated MOMP via type I interferon (IFN) signalling.     

A novel vaccinological evaluation of intranasal vaccine and adjuvant safety for preclinical tests

Vaccines are administered to healthy humans, including infants, so the safety and efficacy must be very high. Therefore, evaluating vaccine safety in preclinical and clinical studies, according to World Health Organization guidelines, is crucial for vaccine development and clinical use. A change in the route of administration is considered to alter a vaccine’s immunogenicity. Several adjuvants have also been developed and approved for use in vaccines. However, the addition of adjuvants to vaccines may cause unwanted immune responses, including facial nerve paralysis and narcolepsy. Therefore, a more accurate and comprehensive strategy must be used to develope next-generation vaccines for ensuring vaccine safety. Previously, we have developed a system with which to evaluate vaccine safety in rats using a systematic vaccinological approach and 20 marker genes. In this study, we developed a safety evaluation system for nasally administered influenza vaccines and adjuvanted influenza vaccines using these marker genes. Expression of these genes increased dose-dependent manner when mice were intranasally administered the toxicity reference vaccine. When the adjuvant CpG K3 or a CpG-K3-combined influenza vaccine was administered intranasally, marker gene expression increased in a CpG-K3-dose-dependent way. A histopathological analysis indicated that marker gene expression correlated with vaccine- or adjuvant-induced phenotypic changes in the lung and nasal mucosa. We believe that the marker genes expression analyses will be useful in preclinical testing, adjuvant development, and selecting the appropriate dose of adjuvant in nasal administration vaccines.     

A therapeutic HIV-1 vaccine reduces markers of systemic immune activation and latent infection in patients under highly active antiretroviral therapy

HIV infection is characterized by chronic immune activation and the establishment of a pool of latently infected cells. Antiretroviral therapy (ART) can suppress viral load to undetectable levels in peripheral blood by standard measure, however immune activation/chronic inflammation and latent infection persist and affect quality of life. We have now shown that a novel therapeutic HIV vaccine consisting of replication-defective HIV (HIVAX), given in the context of viral suppression under ART, can reduce both immune activation/chronic inflammation and latent infection. Immune activation, as measured by percent of CD8 + HLA-DR + CD38 + T cells, approached levels of healthy controls at week 16 following vaccination. Reduced immune activation was accompanied by a reduction in pro-inflammatory cytokines and peripheral α4β7 + plasmacytoid DC (a marker of mucosal immune activation). Levels of both HIV-1 DNA and 2-LTR circles were reduced at week 16 following vaccination, suggesting HIVAX can impact HIV-1 latency and reduce viral replication. Surprisingly, reduced immune activation/chronic inflammation was accompanied by an increase in the percent of memory CD4 + T cells expressing markers PD-1 and TIM-3. In addition, evaluation of HIV-1 Gag-specific CD4 + T cells for expression of 96 T cell related genes pre- and post-therapy revealed increased expression of a number of genes involved in the regulation of immune activation, T cell activation, and antiviral responses. Overall this study provides evidence that vaccination with HIVAX in subjects under long term antiviral suppression can reduce immune activation/chronic inflammation and latent infection (Clinicaltrials.gov, identifier NCT01428596).     

A novel hepatitis B vaccine containing Advax™, a polysaccharide adjuvant derived from delta inulin,induces robust humoral and cellular immunity with minimal reactogenicity in preclinical testing

Although current HBV vaccines have an outstanding record of safety and efficacy, reduced immunogenicity is a problem in those of older age, or having renal impairment or diabetes mellitus. In this study, we tested the ability of Advax™ adjuvant, a novel polysaccharide adjuvant based on delta inulin, to enhance the immunogenicity of hepatitis B surface antigen (HBs) in mice and guinea pigs by comparison to the traditional alum adjuvant. Advax™ provided antigen-sparing, significantly enhanced both anti-HBs antibody titers, and anti-HBs CD4 and CD8 T-cells, with increases in Th1, Th2 and Th17 cytokine responses. Unlike alum, the adjuvant effect of Advax™ was seen even when injected 24 h before the HBs antigen. Advax™ adjuvant similarly enhanced humoral and cellular immune responses in guinea pigs to a third generation preS-HBs antigen. Advax™ adjuvant when combined with HBs antigen could provide enhanced protection over current generation HBV vaccines for immunization of low responder populations.     

Construction and preclinical evaluation of mmCT,a novel mutant cholera toxin adjuvant that can be efficiently produced in genetically manipulated Vibrio cholerae

There is an urgent need for new adjuvants that are effective with mucosally administered vaccines. Cholera toxin (CT) is the most powerful known mucosal adjuvant but is much too toxic for human use. In an effort to develop a useful mucosal adjuvant we have generated a novel non-toxic mutant CT molecule that retains much of the adjuvant activity of native CT. This was achieved by making the enzymatically active A subunit (CTA) recalcitrant to the site-specific proteolytic cleavage (“nicking”) required for toxicity, which was found to require mutations not only in the two residues rendering the molecule resistant to trypsin but also in neighboring sites protecting against cleavage by Vibrio cholerae proteases. This multiple-mutated CT (mmCT) adjuvant protein could be efficiently produced in and purified from the extracellular medium of CT-deleted V. cholerae. The mmCT completely lacked detectable enterotoxicity in an infant mouse model and had >1000-fold reduced cAMP inducing activity compared to native CT in a sensitive mammalian target cell system. It nonetheless proved to have potent adjuvant activity on mucosal and systemic antibody as well as cellular immune responses to mucosally co-administered antigens including oral cholera and intranasal influenza vaccines. We conclude that mmCT is an attractive novel non-toxic mucosal adjuvant for enhancing immune responses to co-administered mucosal vaccines.     

Strong and persistent CD4 T-cell response in healthy adults immunized with a candidate HIV-1 vaccine containing gp120, Nef and Tat antigens formulated in three Adjuvant Systems

This randomized double-blind study aimed to determine the safety and immunogenicity of a gp120/NefTat candidate human immunodeficiency virus type 1 (HIV-1) vaccine formulated with one of three different Adjuvant Systems (AS02_A, AS02_V and AS01_B) in healthy HIV-seronegative adults. All vaccine formulations induced strong HIV-specific CD4⁺ T-cell responses characterized by high lymphoproliferative capacity and IL-2 production that were still detectable 18 months after last immunization, with strongest responses seen in the AS01_B group. Broad coverage was demonstrated against gp120, and to a lesser extent Nef, derived from the most common circulating clades (B, C and circulating recombinant form [CRF]-01). All vaccine formulations exhibited acceptable safety and reactogenicity profiles. The demonstration of superior CD4⁺ T-cell induction by AS01_B provides important guidance for future HIV vaccine development.     

Improved efficacy of DNA vaccination against prostate carcinoma by boosting with recombinant protein vaccine and by introduction of a novel adjuvant epitope

DNA vaccine represents an attractive approach for cancer treatment by inducing active immune-deprivation of gastrin-releasing peptide (GRP) from tumor cells, the growth of which is dependent on the stimulation of GRP. In this study, we developed a DNA vaccine using a plasmid vector to deliver the immunogen of six copies of the B cell epitope GRP_18–27 (GRP6). In order to increase the potency of this DNA vaccine, multiple strategies have been applied including DNA-prime protein-boost immunization and introduction of a foreign T-helper epitope into DNA vaccine. Mice vaccinated DNA vaccine boosting with HSP65-GRP6 protein induced high titer and relatively high avidity of anti-GRP antibodies as well as inhibition effect on the growth of murine prostate carcinoma, superior to the treatment using DNA alone or BCG priming HSP65-GRP6 protein boosting. Furthermore, the introduction of a novel foreign T-helper epitope into the GRP DNA vaccine showed a markedly stronger humoral immune response against GRP and tumor rejection even than the DNA-prime protein-boost strategy. No further stronger immunogenicity of this foreign T-helper epitope modified DNA vaccine was observed even using the strategy of modified DNA vaccine-priming and HSP65-GRP6 boosting method. The data presented demonstrate that improvement of potency of anti-GRP DNA vaccine with the above two feasible approaches should offer useful methods in the development of new DNA vaccine against growth factors for cancer immunotherapy.     

Development of a DNA vaccine expressing a secreted HIV-1 gp41 ectodomain that includes the membrane-proximal external region

A limited number of sites on the HIV-1 Envelope protein are vulnerable to broadly neutralizing antibodies (bnAbs). One of these sites, the membrane proximal external region (MPER), is located at the C-terminus of the gp41 ectodomain (gp41_ecto). This highly conserved sequence is bound by several well-characterized bnAbs. Efforts to produce a gp41 immunogen are in part hampered by the MPER’s hydrophobicity and propensity to induce aggregation. We sought to produce a DNA vaccine expressing a gp41_ecto that is both secreted from mammalian cells and maintains binding by bnAbs to the MPER. Through in silico analysis, we predicted regions of gp41_ecto that could induce aggregation and possibly hinder secretion. We generated deletion mutants of gp41_ecto and tested their ability to be secreted by mammalian cells. Upon deletion of regions in either the fusion peptide (FP) or MPER, secretion of the gp41_ecto increased. In an effort to both augment secretion and maintain binding by bnAbs, we developed constructs with the FP deletion and introduced point mutations in the MPER. Two constructs (gp41 ΔFP and gp41 ΔFP+I682E) maintained binding by gp41 MPER-specific bnAbs (4E10, Z13e1 and 10E8). These were evaluated as DNA vaccines in a mouse model. Both vaccines proved to be immunogenic and appeared to elicit some MPER-specific antibodies that bound gp41 ectodomain-derived proteins but not short peptides spanning the MPER. No neutralizing capacity was detected against a clade C virus containing a homologous MPER.     

Immunization and challenge experiments with a new modified live bovine herpesvirus type 1 marker vaccine prototype adjuvanted with a co-polymer

Western European control programs against bovine herpesvirus type 1 (BoHV-1) infections utilize attenuated BoHV-1 marker vaccines with a deletion of the glycoprotein E (gE) encoding gene. However, a recent study demonstrated the potential risk of virulence recovery of gE-deleted BoHV-1 marker vaccine strains due to recombination (Muylkens et al. [15]). Based on an infectious bacterial artificial chromosome clone, a gE- and thymidine kinase (TK)-gene-deleted BoHV-1 mutant (BoHV-1ΔgEΔTK) was constructed. The recombinant virus was subsequently tested as a novel modified live marker vaccine candidate in an immunization-challenge trial using BoHV-1 seronegative calves. Additionally, a non-virucidal co-polymer was tested together with the recombinant virus acting as a vaccine-adjuvant. Animals were vaccinated twice through intramuscular injection and challenged intranasally 3 weeks later with a virulent BoHV-1 field strain. Duration and titres of challenge virus shedding were significantly reduced in all vaccinees. Importantly, reduction of challenge virus shedding and serological antibody levels in response to vaccination with vaccine preparations containing the co-polymer-adjuvant were markedly improved when compared to vaccine formulations without an adjuvant. Taken together, our study describes a novel double deletion mutant as a safe and efficacious BoHV-1-prototype marker vaccine strain with enhanced protective capacity especially when administered together with a co-polymer adjuvant.     

Influenza vaccine: Development of a novel intranasal and subcutaneous recombinant adjuvant

The synthetic peptide GK-1, derived from Taenia crassiceps, enhances the protection induced by human influenza vaccine in both young and aged mice. Herein, the adjuvant properties of GK-1 fused to the pVIII protein of a heat-inactivated phagemid vector (FGK1) when co-administered with the influenza vaccine were assessed, to evaluate its feasibility as a low-cost adjuvant. In mice, FGK1 significantly increased the expected IgG and IgA anti-influenza antibody levels both in sera and in bronchoalveolar fluids when intranasally or subcutaneously co-administered with influenza vaccine. Single-dose pig co-immunization with FGK1 and influenza vaccine induced serum levels of IgG anti-influenza antibodies similar to those elicited by a two-dose immunization with the influenza vaccine alone. Preclinical evaluation of FGK1 with the influenza vaccine is currently in progress, in order to recommend its use for veterinary purposes.     

A critical analysis of the cynomolgus macaque,Macaca fascicularis,as a model to test HIV-1/SIV vaccine efficacy

The use of a number of non-rhesus macaque species, but especially cynomolgus macaques as a model for HIV-1 vaccine development has increased in recent years. Cynomolgus macaques have been used in the United Kingdom, Europe, Canada and Australia as a model for HIV vaccine development for many years. Unlike rhesus macaques, cynomolgus macaques infected with SIV show a pattern of disease pathogenesis that more closely resembles that of human HIV-1 infection, exhibiting lower peak and set-point viral loads and slower progression to disease with more typical AIDS defining illnesses. Several advances have been made recently in the use of the cynomolgus macaque SIV challenge model that allow the demonstration of vaccine efficacy using attenuated viruses and vectors that are both viral and non-viral in origin. This review aims to probe the details of various vaccination trials carried out in cynomolgus macaques in the context of our modern understanding of the highly diverse immunogenetics of this species with a view to understanding the species-specific immune correlates of protection and the efficacy of vectors that have been used to design vaccines.     

Striking lack of T cell immunodominance in both a multiclade and monoclade HIV-1 epidemic: Implications for vaccine development

Understanding the impact of HIV diversity on immunological responses to candidate immunogens is critical for HIV vaccine development. We investigated the reactivity and immunodominance patterns of HIV-1 consensus group M Gag and Nef in (i) Cameroon, where individuals infected with the predominant CRF02_AG clade were compared with those infected with diverse non-CRF02_AG clades; and (ii) in a multiclade epidemic, namely Cameroon, compared with a monoclade C epidemic, South Africa. We analyzed 57 HIV-infected individuals from Cameroon and 44 HIV-infected individuals from South Africa for differences in detecting HIV-1 consensus M Gag and Nef T cell responses using the IFN-γ ELISpot assay. We found no difference in the predicted epitope coverage between CRF02_AG and non-CRF02_AG viruses for either Gag or Nef. There were no differences in the magnitude and breadth of responses for CRF02_AG and non-CRF02_AG-infected individuals. In contrast, the specificity of epitope targeting was markedly different between the two groups, with fewer than one third (11/38) of peptides commonly recognized in Gag. Furthermore, only one peptide was commonly recognized by at least three individuals from both AG and non-AG groups, indicating poor immunodominance. For Nef, more than half of all targeted peptides (14/27) were recognized by both groups, and four peptides were commonly targeted by at least three individuals. Three times more peptides were exclusively targeted in the diverse non-CRF02_AG group compared to the CRF02_AG group (10 vs. 3). Of note, similar results were obtained when South Africa, a monoclade C epidemic, and Cameroon, a multiclade epidemic, were compared. The central nature of HIV-1 consensus M sequences resulted in their broad recognition, but failed to identify highly immunodominant peptides between homogeneous and diverse HIV epidemics.     

Immunogenicity of a novel engineered HIV-1 clade C synthetic consensus-based envelope DNA vaccine

DNA vaccines require significant engineering in order to generate strong CTL responses in both non-human primates and humans. In this study, we designed a clade C env gene (EY3E1-C) to decrease the genetic distances of virus isolates within clade C and focus the induced T cell responses to conserved clade C epitopes. After generating a consensus sequence by analyzing full-length clade C env early transmitter sequences, several modifications were performed to increase the expression of the EY3E1-C, including codon/RNA optimization, addition of Kozak sequence and addition of an IgE leader sequence. We also shortened the V1 and V2 loops to approximate early transmitter isolate sequences and the cytoplasmic tail was truncated to prevent envelope recycling. When studied as a DNA vaccine in Balb/c mice, compared to a primary codon-optimized clade C envelope DNA vaccine (p96ZM651gp140-CD5), this novel construct is up to three times more potent in driving CTL responses. Importantly this construct not only induces stronger cross-reactive cellular responses within clade C, it also induces stronger immune responses against clade B and group M envelope peptide pools than p96ZM651gp140-CD5. Epitope mapping demonstrated that EY3E1-C was able to induce clade C envelope-specific immune responses against 15 peptide pools, clade B envelope-specific immune responses against 19 peptide pools and group M envelope-specific immune responses against 16 peptide pools out of 29, respectively, indicating that a significant increase in the breadth of induced immune responses. The analysis of antibody responses suggested that vaccination of pEY3E1-C could induce a clade C envelope-specific antibody response. The cellular immune responses of pEY3E1-C could be further enhanced when the DNA was delivered by using electroporation (EP). Thus, the synthetic engineered consensus EY3E1-C gene is capable of eliciting stronger and broader CTL responses than primary clade C envelopes. This finding suggests that such synthetic immunogens could be important for examination of their potential as part of an efficient HIV DNA vaccine.     

Safety and immunogenicity of 23-valent pneumococcal polysaccharide vaccine in HIV-1 infected former drug users

The immunogenicity of 23-valent pneumococcal polysaccharide vaccine was assessed in 57 HIV-1 infected former intravenous drug users and in 20 HIV-1 negative controls. The effect of vaccination on HIV-1 infection was studied in a subgroup of 38 patients, 60% of whom under highly active antiretroviral therapy (HAART). Antibody to capsular polysaccharides from Streptococcus pneumoniae serotypes 3, 4, 6B, 19F, 23 F, and changes in CD4+ count, HIV-1 RNA, proviral DNA and HIV-1 phenotype were measured in pre- and post-vaccination samples.Vaccinations were well-tolerated. The rate of responders was higher (P<0.05) in HIV-1 negative than in HIV-1 infected individuals. No difference in antibody response was found within HIV-1 infected patients stratified according to CD4+ counts. Post-vaccination antibody geometric mean concentrations (GMCs) to the five antigens were higher (P<0.05) than baseline in HIV-1 negative subjects, but not in HIV-1 positive individuals. Those with CD4+ >500 cells/mm(3) showed a significant increase of antibody against type 3 only. Immunisation caused no significant changes in CD4+ counts and in either plasma HIV-1 RNA nor proviral DNA levels. Pneumococcal vaccination does not induce virological or immunological deterioration in HIV infected patients, but the antibody response to a single dose of vaccine is poor.     

Boosters of a therapeutic HIV-1 vaccine induce divergent T cell responses related to regulatory mechanisms

Therapeutic human immunodeficiency virus (HIV) vaccines aim to reduce disease progression by inducing HIV-specific T cells. Vacc-4x are peptides derived from conserved domains within HIV-1 p24 Gag. Previously, Vacc-4x induced T cell responses in 90% of patients which were associated with reduced viral loads. Here we evaluate the effects of Vacc-4x boosters on T cell immunity and immune regulation seven years after primary immunization. Twenty-five patients on effective antiretroviral therapy received two Vacc-4x doses four weeks apart and were followed for 16 weeks. Vacc-4x T cell responses were measured by proliferation (CFSE), INF-γ, CD107a, Granzyme B, Delayed-Type Hypersensitivity test (DTH) and cytokines and chemokines (Luminex). Functional regulation of Vacc-4x-specific T cell proliferation was estimated in vitro using anti-IL-10 and anti-TGF-ß monoclonal antibodies.     

Innate immunemodulator containing adjuvant formulated HA based vaccine protects mice from lethal infection of highly pathogenic avian influenza H5N1 virus

The highly pathogenic avian influenza (HPAI) H5N1 viruses and their spillover into the human population pose substantial economic and public health threats. Although antiviral drugs have some effect in treating influenza infection, vaccination is still the most effective intervention to prevent possible pandemic outbreaks. We have developed a novel H5 influenza vaccine to improve the world’s pandemic preparedness. We produced a hemagglutinin (HA) of HPAI H5N1 virus A/Alberta/01/2014 (AB14) using both mammalian (m) and bacterial (b) expression systems. The purified recombinant proteins were formulated with a proprietary adjuvant (TriAdj) and their efficacy as vaccine candidates was evaluated in mice. Intramuscular delivery of two doses of TriAdj formulated mammalian expressed HA (m-HA/TriAdj) was shown to provide full protection against a lethal challenge of AB14 in mice. In contrast, bacterially expressed HA with TriAdj (b-HA/TriAdj), b-HA without adjuvant, and m-HA without adjuvant resulted in no protection in immunized mice. Furthermore, m-HA/TriAdj elicited significantly higher levels of balanced Th1 and Th2 responses and neutralizing antibody titres. All the mice in the m-HA/TriAdj group survived a lethal AB14 H5N1 challenge and showed no signs of disease or infection as demonstrated by no loss of body weight or detectable virus in the lungs. Our results suggest that m-HA formulated with TriAdj has potential to protect against pandemic H5N1 in the event of its cross over to the human host.     

Effect of different adjuvant formulations on the immunogenicity and protective effect of a live Mycoplasma hyopneumoniae vaccine after intramuscular inoculation

Mycoplasma hyopneumoniae (M. hyopneumoniae) vaccine strain 168 is an intrapulmonically injected attenuated live vaccine that is available in the Chinese market. The aim of this study was to develop suitable adjuvants for this live vaccine to provide effective protection after intramuscular inoculation. Several adjuvant components were screened to assess their toxicity for the live vaccine, and various adjuvant formulations were then designed and prepared. Vaccines supplemented with these adjuvants were used to immunize mice intramuscularly to assess the capacity of the adjuvants to induce a specific immune response. The screened formulations were then evaluated in pigs. Seven of the eight adjuvant components did not affect the viability of the live vaccine, and seven different adjuvant formulations were then designed. In mice, the ISCOM-matrix adjuvant and the levamisole–chitosan mixture adjuvant significantly enhanced serum IgG responses against M. hyopneumoniae, while lymphocyte proliferation was enhanced by the ISCOM-matrix adjuvant, the carbomer–astragalus polysaccharide mixture adjuvant and an oil-in-water emulsion adjuvant. These four adjuvants were evaluated in pigs. Enhancement of specific lymphocyte proliferation responses was observed in the groups vaccinated with the ISCOM-matrix adjuvant and the carbomer–astragalus polysaccharide mixture adjuvant. Significant enhancement of serum IgG antibody production was observed before challenge in pigs vaccinated with the carbomer–astragalus polysaccharide mixture adjuvant and the levamisole–chitosan mixture adjuvant, while after challenge, all of the animals that received vaccines containing adjuvants had higher antibody concentrations against M. hyopneumoniae than unvaccinated animals. Animals inoculated with a vaccine containing the ISCOM-matrix adjuvant (median score 3.57) or the carbomer–astragalus polysaccharide mixture adjuvant (median score 5.28) had reduced lesion scores compared to unvaccinated animals (median score 14.81). These studies will help in the development of appropriate adjuvants for intramuscular administration of this live M. hyopneumoniae vaccine.