Intramuscular Matrix-M-adjuvanted virosomal H5N1 vaccine induces high frequencies of multifunctional Th1 CD4 cells and strong antibody responses in mice

Ideally, a candidate pandemic influenza vaccine should elicit rapid and strong cell-mediated and humoral immune responses, which are long-lasting and exhibit broad cross-reactivity against drifted strains. The present study investigated the detailed humoral and cellular immune responses in mice vaccinated intranasally or intramuscularly with inactivated influenza H5N1 (NIBRG-14) virosomal vaccine alone or formulated with Matrix-M adjuvant. The intramuscular Matrix-M-adjuvanted vaccine induced a strong immediate and long-term humoral immune response with high cross-reactivity against drifted H5N1 viruses and showed a dose-sparing potential. Additionally, the vaccine induced a balanced Th1/Th2 cytokine profile and most importantly high frequencies of multifunctional Th1 CD4⁺ cells. Our results highlight that Matrix-M adjuvant is a promising parenteral adjuvant for formulating pandemic candidate vaccines.     

Humoral and cellular immune responses to split-virion H5N1 influenza vaccine in young and elderly adults

We evaluated the humoral and cellular immunogenicity of adjuvanted and non-adjuvanted H5N1 influenza vaccine in two groups of 300 adults: aged 18–60 and >60 years in a randomized, open-label, uncontrolled phase 2 trial. Participants received two injections (D0, D21) of 7.5 μg hemagglutinin without adjuvant or 30 μg with aluminum hydroxide adjuvant. Antibody responses and cytokine secretion were assessed before and after vaccination. Excluding the 6/300 non-elderly and 47/300 elderly participants with pre-existing antibodies, geometric mean titers (dil⁻¹) on D42 were higher with 30 μg+Ad and were comparable between age groups. Participants with pre-existing antibodies responded strongly to the first vaccination (GMTs in the range 147–228 on D21). Vaccination increased both Th1 and Th2 T-cell responses. The predominantly Th1 profile observed before vaccination was unaffected by vaccination. H5N1 influenza vaccine is no less immunogenic in elderly adults than in younger adults and, due to a higher proportion non-naïve elderly, immunogenicity was higher in this latter group.     

A universal epitope-based influenza vaccine and its efficacy against H5N1

Previous studies have shown that a recombinant vaccine expressing four highly conserved influenza virus epitopes has a potential for a broad spectrum, cross-reactive vaccine; it induced protection against H1, H2 and H3 influenza strains. Here, we report on the evaluation of an epitope-based vaccine in which six conserved epitopes, common to many influenza virus strains are expressed within a recombinant flagellin that serves as both a carrier and adjuvant. In an HLA-A2.1 transgenic mice model, this vaccine induced both humoral and cellular responses and conferred some protection against lethal challenge with the highly pathogenic H5N1 avian influenza strain. Hence, it is expected to protect against future strains as well. The data presented, demonstrate the feasibility of using an array of peptides for vaccination, which might pave the way to an advantageous universal influenza virus vaccine that does not require frequent updates and/or annual immunizations.     

A novel adjuvant G3 induces both Th1 and Th2 related immune responses in mice after immunization with a trivalent inactivated split-virion influenza vaccine

A preferred adjuvant should promote both Th1 and Th2 responses. However, most adjuvants in common use are biased towards a Th2-driven response. Therefore, the ability of a novel saponin-based adjuvant G3 to inducing balanced Th1 and Th2 responses in BALB/c mice immunized with a split trivalent seasonal influenza vaccine was evaluated in comparison to that of the adjuvant Al(OH)₃. Clear differences in the IgG profiles induced by G3, Al(OH)₃ or non-adjuvanted vaccine were recorded. Both adjuvants enhanced high and similar levels of the Th2 associated IgG1 subtype compared to mice given vaccine alone. Only G3 enhanced the IgG2a subclass reflecting a Th1 response, whereas Al(OH)₃ even abrogated the IgG2a production. Accordingly, G3 enhanced the production of IL-2 and IFN-γ and also of IL-2/IFN-γ double secreting cells, emphasizing the strong Th1 driving effect of G3. Only Al(OH)₃ increased splenocyte production of IL-17. Taken together, the results indicate a strong propensity for G3 to induce both Th1 and Th2 driven immune responses.     

Alum-adjuvanted H5N1 whole virion inactivated vaccine (WIV) induced IgG1 and IgG4 antibody responses in young children

IgG subclass antibody responses are not fully understood. Alum-adjuvanted H5N1whole virion inactivated vaccine (WIV), a genetically reassortant vaccine seed strain originating from H5N1/A/Vietnam/1194/2004 and PR-8, induced significantly stronger antibody responses in neutralizing antibodies in children. In this report, IgG subclass antibody responses were investigated, and most serum samples were positive for IgG1 antibody before immunization. A significant response (more than 4-fold increase) of IgG1 antibody was observed in 67/193 (34.7%) and that of gG4 antibodies in 42/193(21.8%). Children <4 years of age showed a significant increase in IgG subclass antibodies but those ≥4 years showed lower responses. Alum- adjuvanted H5N1WIV induced an efficient immune response in young children especially <4 years.     

A new adjuvanted nanoparticle-based H1N1 influenza vaccine induced antigen-specific local mucosal and systemic immune responses after administration into the lung

Annually influenza virus infections are responsible for hospitalization and mortality, especially in high risk groups. Constant antigenic changes in seasonal influenza viruses resulted from antigenic shifts and antigenic drifts, enable emerging of novel virus subtypes that may reduce current vaccine efficacy and impose the continuous revision of vaccine component. Currently available vaccines are usually limited by their production processes in terms of rapid adaptation to new circulating subtypes in high quantities meeting the global demand. Thus, new approaches to rapidly manufacture high yields of influenza vaccines are required. New technologies to reach maximal protection with minimal vaccine doses also need to be developed.     

Chimpanzee adenovirus vector-based avian influenza vaccine completely protects mice against lethal challenge of H5N1

Highly pathogenic avian H5N1 viruses may give rise to the next influenza pandemic due to their reassortment and mutation of the genome. Vaccine against this virus is important for coping with its potential threat. Chimpanzee adenovirus (Ad) vectors are a novel type of vaccine vectors that share the advantages of human serotype Ad vectors but without being affected by pre-existing human neutralizing antibody to the vaccine vector. Based on a replication-deficient chimpanzee Ad vector, AdC7, we generated a novel H5N1 vaccine candidate AdC7-H5HA that expresses H5N1 Hemagglutinin (HA). When tested in mice, the vaccine significantly reduced the virus load and pathological lesions in the lung tissues, and conferred complete protection against lethal challenge by a homologous virus. Mechanistically, the AdC7-H5HA vaccine can induce both HA-specific humoral and cell-mediated immune responses in mice. Also, sera transfer experiments demonstrated that neutralizing antibodies alone could provide protection. In conclusion, our results show that chimpanzee Ad vector expressing influenza virus HA may represent a promising vaccine candidate for H5N1 viruses and other influenza virus subtypes.     

Recombinant baculovirus vaccine containing multiple M2e and adjuvant LTB induces T cell dependent,cross-clade protection against H5N1 influenza virus in mice

H5N1, highly pathogenic avian influenza poses, a threat to animal and human health. Rapid changes in H5N1 viruses require periodic reformulation of the conventional strain-matched vaccines, thus emphasizing the need for a broadly protective influenza vaccine. Here, we constructed BV-Dual-3M2e-LTB, a recombinant baculovirus based on baculovirus display and BacMam technology. BV-Dual-3M2e-LTB harbors a gene cassette expressing three tandem copies of the highly conserved extracellular domain of influenza M2 protein (M2e) and the mucosal adjuvant, LTB. We showed that BV-Dual-3M2e-LTB displayed the target protein (M2e/LTB) on the baculoviral surface and expressed it in transduced mammalian cells. BV-Dual-3M2e-LTB, when delivered nasally in mice, was highly immunogenic and induced superior levels of anti-M2e IgA than the non-adjuvanted baculovirus (BV-Dual-3M2e). Importantly, after challenge with different H5N1 clades (clade 0, 2.3.2.1, 2.3.4 and 4), mice inoculated with BV-Dual-3M2e-LTB displayed improved survival and decreased lung virus shedding compared with mice inoculated with BV-Dual-3M2e. The enhanced protection from BV-Dual-3M2e-LTB is mediated by T cell immunity and is primarily based on CD8⁺ T cells, while mucosal antibodies alone were insufficient for protection from lethal H5N1 challenge. These results suggest that BV-Dual-3M2e-LTB has potential to protect against a broad range of H5N1 strains thereby providing a novel direction for developing broadly protective vaccines based on cellular immunity.     

Feasibility of single-shot H5N1 influenza vaccine in ferrets, macaques and rabbits

The feasibility of a single-shot, low-dose vaccination against pandemic influenza was investigated. The immunogenicity and safety of whole inactivated, cell culture-derived H5N1 virus plus CoVaccine HT™ as adjuvant was tested in various animal species. In ferrets, doses of 4.0 and 7.5 μg H5N1 (NIBRG-14; A/Vietnam/1194/04; clade 1) without adjuvant gave low geometric mean haemagglutination inhibition (HI) titres (GMTs) of 21–65 three weeks after intramuscular (IM) injection. The addition of 0.25–4 mg CoVaccine HT™ resulted in GMTs of 255–1470 corresponding with 4–25-fold increases. A second immunization caused GMTs of 8914–23,525 two weeks later, which confirmed strong priming. One out of 8 ferrets injected with antigen alone and 5 out of 32 ferrets injected with adjuvanted H5N1 demonstrated minimal transient, local reactions and two animals immunized with adjuvanted H5N1 exhibited increased body temperature one day after injection. In macaques, 5 μg H5N1 with CoVaccine HT™ or aluminium hydroxide as adjuvant elicited GMTs of 172 and 11, respectively three weeks later. A second immunization resulted in GMTs of 1751 and 123, respectively four weeks later. Analysis of cross-reactivity of antibodies after the first immunization with NIBRG-14 adjuvanted plus CoVaccine HT™ revealed GMTs of 69 against NIBRG-23 (A/turkey/Turkey/1/05; clade 2.2) and 42 against IBCDC-RG-2 (A/Indonesia/5/05-like; clade 2.1.3) while titres with aluminium hydroxide were <10. After the second immunization with CoVaccine HT™, GMT against NIBRG-23 was 599 and against IBCDC-RG-2 254, while those with aluminium hydroxide were 23 and 13, respectively. No local or systemic adverse events were detected in macaques. Safety of 5 μg H5N1 plus 0, 2 or 4 mg CoVaccine HT™ was investigated in a repeated dose study in rabbits. Groups of 6 or 9 male and female animals were immunized IM three times at three week intervals. None of the animals exerted treatment-related adverse reactions during the study or at necropsy 3 or 4 days after treatment.     

Nasal delivery of Protollin-adjuvanted H5N1 vaccine induces enhanced systemic as well as mucosal immunity in mice

Sporadic, yet frequent human infections with avian H5N1 influenza A viruses continue to pose a potential pandemic threat. Poor immunogenicity of unadjuvanted H5N1 vaccines warrants developing novel adjuvants and formulations as well as alternate delivery systems to improve their immunogenicity and efficacy. Here, we show that Protollin, a nasal adjuvant composed of Neisseria meningitides outer membrane proteins non-covalently linked to Shigella flexneri 2a lipopolysaccharide, is a potent nasal adjuvant for an inactivated split virion H5N1 clade 1 A/Viet Nam1203/2004 (A/VN/1203/04) vaccine in a mouse model. Protollin-adjuvanted vaccines elicited enhanced serum protective hemagglutination inhibition titers, mucosal IgA responses, and H5N1-specific cell-mediated immunity that resulted in complete protection against a lethal challenge with a homologous virus as well as a heterologous clade 2 virus A/Indonesia/05/2005 (A/IN/05/05). Detailed analysis of adaptive immunity revealed that Protollin increased the frequency of lymphoid- as well as local tissue-resident antibody-secreting cells, local germinal center reaction of B cells, broad-spectrum of CD4 T cell response. Our findings suggest that nasal delivery of H5N1 vaccine with Protollin adjuvant can overcome the poor immunogenicity of H5N1 vaccines, induce both cellular and humoral immune responses, enhance protection against challenge with clade 1 and clade 2 H5N1 viruses and achieve significant antigen dose-sparing.     

The novel adjuvant CoVaccineHT™ increases the immunogenicity of cell-culture derived influenza A/H5N1 vaccine and induces the maturation of murine and human dendritic cells in vitro

A candidate influenza H5N1 vaccine based on cell-culture-derived whole inactivated virus and the novel adjuvant CoVaccineHT™ was evaluated in vitro and in vivo. To this end, mice were vaccinated with the whole inactivated influenza A/H5N1 virus vaccine with and without CoVaccineHT™ and virus-specific antibody and cellular immune responses were assessed. The addition of CoVaccineHT™ increased virus specific primary and secondary antibody responses against the homologous and an antigenically distinct heterologous influenza A/H5N1 strain. The superior antibody responses induced with the CoVaccineHT™-adjuvanted vaccine correlated with the magnitude of the virus-specific CD4+ T helper cell responses. CoVaccineHT™ did not have an effect on the magnitude of the CD8+ T cell response. In vitro, CoVaccineHT™ upregulated the expression of co-stimulatory molecules both on mouse and human dendritic cells and induced the secretion of pro-inflammatory cytokines TNF-α, IL-6, IL-1β and IL-12p70 in mouse- and IL-6 in human dendritic cells. Inhibition experiments indicated that the effect of CoVaccineHT™ is mediated through TLR4 signaling. These data suggest that CoVaccineHT™ also will increase the immunogenicity of an influenza A/H5N1 vaccine in humans.     

Single dose of oil-adjuvanted inactivated vaccine protects chickens from lethal infections of highly pathogenic H5N1 influenza virus

The highly pathogenic H5N1 influenza viruses are endemic in poultry in many countries, but continuously infect humans and cause human mortality. H5N1 influenza viruses have been regarded as a pandemic candidate. In a pandemic event by this virus, the protection of poultry with an effective vaccine will help to greatly reduce the spread of this virus to humans since it easily infects poultry. Here we showed that immunization with one dose of oil-adjuvanted inactivated H5N1 vaccine could protect chickens from lethal infection by highly pathogenic H5N1 influenza virus until 12 weeks post-immunization. The complete protection of chickens depended on the amount of HA antigens in the vaccine. Complete homologous protection required over 1.25 μg of HA antigens and complete heterologous protection required over 5.0 μg of HA antigens. The bivalent H5N1 inactivated vaccine composed of 1.25 μg of each antigen from clade 1 and clade 2.3.4 H5N1 influenza virus completely protected chickens from the lethal challenge of both viruses. When we determined the induction of antibody subtypes in tissues including nasal cavity, trachea, and lungs, the IgG subtype of antibody was induced more than the IgM or IgA subtype of antibody. Taken together, our results suggest that one dose of oil-adjuvanted inactivated H5N1 vaccine could provide chickens with sterile immunity against the homologous highly pathogenic H5N1 influenza virus.     

Protection of pregnant mice,fetuses and neonates from lethality of H5N1 influenza viruses by maternal vaccination

The highly pathogenic H5N1 influenza viruses are one of candidates for the next pandemic. Information on protective immunity for pregnant animals by vaccination against the H5N1 influenza virus is limited. Here, we show that the immunization of pregnant mice with inactivated H5N1 influenza vaccine protects them, their fetuses, and their infant mice from H5N1 influenza viruses. Pregnant mice immunized with two doses of H5N1 influenza vaccine were protected from homologous infections of H5N1 influenza viruses with no viruses detected in fetuses, and that they were protected upto 30% from heterologous infections of H5N1 influenza viruses with viruses detected in fetuses. The infant mice born to mothers immunized with H5N1 influenza vaccine were fully protected from infections of H5N1 influenza viruses for upto 4 weeks of age. The protection of infant mice was closely related to the presence of IgG2a antibody in lung, heart, and rectum tissues. Our results suggest that maternal vaccination may be critical for protecting pregnant animals, their fetuses, and their infant mice from lethal infections of H5N1 influenza viruses.     

Inactivated trivalent seasonal influenza vaccine induces limited cross-reactive neutralizing antibody responses against 2009 pandemic and 1934 PR8 H1N1 strains

Background

In June 2009, we conducted a prospective study in Singapore on 51 individuals to determine their serologic responses before and following receipt of the 2009 Southern Hemisphere seasonal influenza vaccine.

Materials and methods

Paired serum samples were obtained before and 3–4 weeks after vaccination. Virus microneutralization assays were performed to quantify antibodies against A/Brisbane/59/2007 vaccine, pandemic H1N1-2009 and A/Puerto Rico/08/34 H1N1 strains.

Results

Post-vaccination, 43%, 12% and 24% of subjects displayed a 4-fold or greater rise in neutralizing antibody titers against the three strains, respectively. There was a positive correlation among individuals who showed increased titers to both pandemic H1N1-2009 and A/Puerto Rico/08/34 (p < 0.001). However, this correlation was not observed for A/Brisbane/59/2007 with either strain. The relative conservation and accessibility of predicted B-cell epitopes may explain the limited cross-reactivity of the antibodies directed against common H1N1 epitopes.

Conclusions

These results suggest that seasonal influenza vaccination confers a certain degree of cross-protection to other H1N1 strains. The correlation in cross-reactive antibody titers to A/Puerto Rico/08/34 and pandemic H1N1-2009 implies that previous exposure to pre-1957 H1N1 strains may confer some protection against the 2009 pandemic strain.     

Expression of H5 hemagglutinin vaccine antigen in common duckweed (Lemna minor) protects against H5N1 high pathogenicity avian influenza virus challenge in immunized chickens

A synthetic hemagglutinin (HA) gene from the highly pathogenic avian influenza (HPAI) virus A/chicken/Indonesia/7/2003 (H5N1) (Indo/03) was expressed in aquatic plant Lemna minor (rLemna-HA). In Experiment 1, efficacy of rLemna-HA was tested on birds immunized with 0.2 μg or 2.3 μg HA and challenged with 10⁶ mean chicken embryo infectious doses (EID₅₀) of homologous virus strain. Both dosages of rLemna-HA conferred clinical protection and dramatically reduced viral shedding. Almost all the birds immunized with either dosage of rLemna-HA elicited HA antibody titers against Indo/03 antigen, suggesting an association between levels of anti-Indo/03 antibodies and protection. In Experiment 2, efficacy of rLemna-HA was tested on birds immunized with 0.9 μg or 2.2 μg HA and challenged with 10⁶ EID₅₀ of heterologous H5N1 virus strains A/chicken/Vietnam/NCVD-421/2010 (VN/10) or A/chicken/West Java/PWT-WIJ/2006 (PWT/06). Birds challenged with VN/10 exhibited 100% survival regardless of immunization dosage, while birds challenged with PWT/06 had 50% and 30% mortality at 0.9 μg HA and 2.2 μg HA, respectively. For each challenge virus, viral shedding titers from 2.2 μg HA vaccinated birds were significantly lower than those from 0.9 μg HA vaccinated birds, and titers from both immunized groups were in turn significantly lower than those from sham vaccinated birds. Even if immunized birds elicited HA titers against the vaccine antigen Indo/03, only the groups challenged with VN/10 developed humoral immunity against the challenge antigen. None (rLemna-HA 0.9 μg HA) and 40% (rLemna-HA 2.2 μg HA) of the immunized birds challenged with PWT/06 elicited pre-challenge antibody titers, respectively. In conclusion, Lemna-expressed HA demonstrated complete protective immunity against homologous challenge and suboptimal protection against heterologous challenge, the latter being similar to results from inactivated whole virus vaccines. Transgenic duckweed-derived HA could be a good alternative for producing high quality antigen for an injectable vaccine against H5N1 HPAI viruses.     

Avian influenza H5N1 in naturally infected domestic cat

We report H5N1 virus infection in a domestic cat infected by eating a pigeon carcass. The virus isolated from the pigeon and the cat showed the same cluster as the viruses obtained during the outbreak in Thailand. Since cats are common house pets, concern regarding disease transmission to humans exists.     

Highly pathogenic H5N1 influenza virus in smuggled Thai eagles, Belgium

We report the isolation and characterization of a highly pathogenic avian influenza A/H5N1 virus from Crested Hawk-Eagles smuggled into Europe by air travel. A screening performed in human and avian contacts indicated no dissemination occurred. Illegal movements of birds are a major threat for the introduction of highly pathogenic avian influenza.     

Cold-adapted X-31 live attenuated 2009 pandemic H1N1 influenza vaccine elicits protective immune responses in mice and ferrets

The 2009 pandemic influenza H1N1 (pdmH1N1) is characterized by rapid transmission among humans and disproportionate infection to children and young adults. Although the pdmH1N1 demonstrated less lethality than initially expected and has now moved into its post-pandemic period, it remains highly possible that through antigenic shift or antigenic drift the pdmH1N1 might re-emerge in the future as a more virulent strain than before, underscoring the need for vaccination prior to an outbreak. Using X-31 ca as a backbone strain, we generated a live attenuated pdmH1N1 vaccine and evaluated its potential as a safe and effective vaccine using mouse and ferret models. Despite an acceptable level of attenuation phenotypes, single dose of immunization with the vaccine efficiently stimulated both systemic and mucosal antibody responses and provided complete protection against lethal challenge with wild type pdmH1N1 virus, even at the lowest immunization dose of 10³ PFU. The promising results of safety, immunogenicity, and protective efficacy of the vaccine not only contribute to expanding the repertoire of live vaccines as a judicious choice for pandemic H1N1 preparedness, but also suggest the great potential of X-31 ca donor strain to serve as reliable platform for generating diverse live vaccine constructs against seasonal influenza viruses and other pandemic strains.     

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 recent seasonal influenza vaccination on serum antibody responses to candidate pandemic influenza A/H5N1 vaccines: A meta-analysis

Recent studies have suggested that among those receiving seasonal influenza vaccine (SIV), reduced immunogenicity is observed in recently vaccinated (RV; within the past season or 2) persons when compared with those not recently vaccinated (NRV). We performed a meta-analysis to assess the effect of recent immunization with SIV on serum H5 hemagglutination inhibition (HAI) antibody responses after influenza A/H5N1 vaccination using data from a series of randomized controlled trials. The primary outcome was seroconversion measured by HAI assays following receipt of 2 doses of H5N1 vaccine. The geometric mean titer (GMT) of serum HAI antibody after vaccination was the secondary outcome. Analyses were performed using propensity score (PS) matching. The PS for each individual in the meta-analysis cohort was calculated using logistic regression and covariates included age, gender, race, antigen dose, adjuvant, statin use and vaccine manufacturer. 2015 subjects enrolled in 7 clinical trials were eligible for inclusion in the meta-analysis cohort; among these, 915 (45%) were RV. 901 RV subjects were matched (1:1) with replacement to a subject who was NRV. Subjects who received SIV within the previous season were significantly less likely to seroconvert following H5N1 vaccination (adjusted odds ratio 0.76; 95%CI 0.60–0.96; p = 0.024), and the GMT was 18% higher among NRV subjects (GM ratio of HAI antibody 1.18; 95%CI 1.04–1.33; p = 0.008). Further work is needed to better define the effects of, and mechanisms contributing to, reduced immune responses to H5N1 vaccine among RV subjects.