Single HA2 mutation increases the infectivity and immunogenicity of a live attenuated H5N1 intranasal influenza vaccine candidate lacking NS1.

BACKGROUND: H5N1 influenza vaccines, including live intranasal, appear to be relatively less immunogenic compared to seasonal analogs. The main influenza virus surface glycoprotein hemagglutinin (HA) of highly pathogenic avian influenza viruses (HPAIV) was shown to be more susceptible to acidic pH treatment than that of human or low pathogenic avian influenza viruses. The acidification machinery of the human nasal passageway in response to different irritation factors starts to release protons acidifying the mucosal surface (down to pH of 5.2). We hypothesized that the sensitivity of H5 HA to the acidic environment might be the reason for the low infectivity and immunogenicity of intranasal H5N1 vaccines for mammals. METHODOLOGY/PRINCIPAL FINDINGS: We demonstrate that original human influenza viruses infect primary human nasal epithelial cells at acidic pH (down to 5.4), whereas H5N1 HPAIVs lose infectivity at pH ≤ 5.6. The HA of A/Vietnam/1203/04 was modified by introducing the single substitution HA2 58K→I, decreasing the pH of the HA conformational change. The H5N1 reassortants containing the indicated mutation displayed an increased resistance to acidic pH and high temperature treatment compared to those lacking modification. The mutation ensured a higher viral uptake as shown by immunohistochemistry in the respiratory tract of mice and 25 times lower mouse infectious dose50. Moreover, the reassortants keeping 58K→I mutation designed as a live attenuated vaccine candidate lacking an NS1 gene induced superior systemic and local antibody response after the intranasal immunization of mice. CONCLUSION/SIGNIFICANCE: Our finding suggests that an efficient intranasal vaccination with a live attenuated H5N1 virus may require a certain level of pH and temperature stability of HA in order to achieve an optimal virus uptake by the nasal epithelial cells and induce a sufficient immune response. The pH of the activation of the H5 HA protein may play a substantial role in the infectivity of HPAIVs for mammals.

Mutations affecting the stability of the haemagglutinin molecule impair the immunogenicity of live attenuated H3N2 intranasal influenza vaccine candidates lacking NS1.

The isolation and cultivation of human influenza viruses in embryonated hen eggs or cell lines often leads to amino acid substitutions in the haemagglutinin (HA) molecule. We found that the propagation of influenza A H3N2 viruses on Vero cells may trigger the appearance of HA destabilising mutations, affecting viral resistance to low pH or high temperature treatment. Two ΔNS1 reassortants, containing the HA sequences identical to the original human H3N2 influenza virus isolates were constructed. Passages of these viruses on Vero cells led to the appearance of single mutations in the HA(1) L194P or HA(2) G75R subunits that impaired virus stability. The original HA sequences and the stable phenotypes of the primary isolates were preserved if reassortants were passaged by infection at pH 5.6 and cultivation in medium at pH 6.5. Corresponding ΔNS1 reassortants were compared for their immunogenicity in ferrets upon intranasal immunisation. Vaccine candidates containing HA mutations demonstrated significantly lower immunogenicity compared to those without mutations. Thus, the retaining of the original HA sequences of human viruses during vaccine production might be crucial for the efficacy of live attenuated influenza vaccines.

A novel type of influenza vaccine: safety and immunogenicity of replication-deficient influenza virus created by deletion of the interferon antagonist NS1.

BACKGROUND. The nonstructural protein NS1 of influenza virus counteracts the interferon-mediated immune response of the host. By deleting the open reading frame of NS1, we have generated a novel type of influenza vaccine. We studied the safety and immunogenicity of an influenza strain lacking the NS1 gene (DeltaNS1-H1N1) in healthy volunteers. METHODS. Healthy seronegative adult volunteers were randomized to receive either a single intranasal dose of the DeltaNS1-H1N1 A/New Caledonia vaccine at 1 of 5 dose levels (6.4, 6.7, 7.0, 7.4, and 7.7 log(10) median tissue culture infective dose) (n = 36 recipients) or placebo (n = 12 recipients). RESULTS. Intranasal vaccination with the replication-deficient DeltaNS1-H1N1 vaccine was well tolerated. Rhinitis-like symptoms and headache were the most common adverse events identified during the 28-day observation period. Adverse events were similarly distributed between the treatment and placebo groups. Vaccine-specific local and serum antibodies were induced in a dose-dependent manner. In the highest dose group, vaccine-specific antibodies were detected in 10 of 12 volunteers. Importantly, the vaccine also induced neutralizing antibodies against heterologous drift variants. CONCLUSIONS. We show that vaccination with an influenza virus strain lacking the viral interferon antagonist NS1 induces statistically significant levels of strain-specific and cross-neutralizing antibodies despite the highly attenuated replication-deficient phenotype. Further studies are warranted to determine whether these results translate into protection from influenza virus infection. TRIAL REGISTRATION. ClinicalTrials.gov identifier: NCT00724997 .

Preclinical evaluation of a replication-deficient intranasal DeltaNS1 H5N1 influenza vaccine.

BACKGROUND: We developed a novel intranasal influenza vaccine approach that is based on the construction of replication-deficient vaccine viruses that lack the entire NS1 gene (DeltaNS1 virus). We previously showed that these viruses undergo abortive replication in the respiratory tract of animals. The local release of type I interferons and other cytokines and chemokines in the upper respiratory tract may have a "self-adjuvant effect", in turn increasing vaccine immunogenicity. As a result, DeltaNS1 viruses elicit strong B- and T- cell mediated immune responses. METHODOLOGY/PRINCIPAL FINDINGS: We applied this technology to the development of a pandemic H5N1 vaccine candidate. The vaccine virus was constructed by reverse genetics in Vero cells, as a 5:3 reassortant, encoding four proteins HA, NA, M1, and M2 of the A/Vietnam/1203/04 virus while the remaining genes were derived from IVR-116. The HA cleavage site was modified in a trypsin dependent manner, serving as the second attenuation factor in addition to the deleted NS1 gene. The vaccine candidate was able to grow in the Vero cells that were cultivated in a serum free medium to titers exceeding 8 log(10) TCID(50)/ml. The vaccine virus was replication deficient in interferon competent cells and did not lead to viral shedding in the vaccinated animals. The studies performed in three animal models confirmed the safety and immunogenicity of the vaccine. Intranasal immunization protected ferrets and mice from being infected with influenza H5 viruses of different clades. In a primate model (Macaca mulatta), one dose of vaccine delivered intranasally was sufficient for the induction of antibodies against homologous A/Vietnam/1203/04 and heterologous A/Indonesia/5/05 H5N1 strains. CONCLUSION/SIGNIFICANCE: Our findings show that intranasal immunization with the replication deficient H5N1 DeltaNS1 vaccine candidate is sufficient to induce a protective immune response against H5N1 viruses. This approach might be attractive as an alternative to conventional influenza vaccines. Clinical evaluation of DeltaNS1 pandemic and seasonal influenza vaccine candidates are currently in progress.

Development of a live-attenuated influenza B DeltaNS1 intranasal vaccine candidate.

We discovered a unique, single amino acid mutation in the influenza B M1 protein promoting viral growth of NS1 truncation mutants in Vero cells. Due to this mutation, we were able to generate an influenza B virus lacking the complete NS1 open reading frame (DeltaNS1-B virus) by reverse genetics, which was growing to titers of 8log(10)TCID(50)/ml in a Vero cell culture-based micro-carrier fermenter. The DeltaNS1-B vaccine candidate was attenuated in IFN-competent hosts such as human alveolar epithelial cells (A549) similar to influenza A DeltaNS1 viruses. In ferrets, the DeltaNS1-B virus was replication-deficient and did not provoke any clinical symptoms. Importantly, a single intranasal immunization of ferrets at a dose as low as 6 log(10)TCID(50)/animal induced a significant HAI response and provided protection against challenge with wild-type influenza B virus. So far, the lack of a DeltaNS1-B virus component growing to high titers in cell culture has been limiting the possibility to formulate a trivalent vaccine based on deletion of the NS1 gene. Our study closes this gap and paves the way for the clinical evaluation of a seasonal, trivalent, live replication-deficient DeltaNS1 intranasal influenza vaccine.

Structure and function of YcnD from Bacillus subtilis, a flavin-containing oxidoreductase.

YcnD from the gram-positive bacterium Bacillus subtilis is a member of a family of bacterial proteins that act as NADH- and/or NADPH-dependent oxidoreductases. Here, we report for the first time on the biochemical characterization of the purified protein, demonstrating that YcnD is an FMN-containing enzyme that can be reduced by NADH or NADPH (Km = 6.4 and 4.4 microM, respectively). In the presence of free FMN as the electron-accepting substrate, the latter reductant showed a ping-pong Bi-Bi reaction mechanism, whereas utilization of NADH is competitively inhibited by this substrate. This finding suggests that NADPH is the physiological reductant of the enzyme. We also show that YcnD reduces nitro-organic compounds, chromate, and a series of azo dyes. The reduction of azo dyes appears to be mediated by free reduced FMN because the reaction is considerably slower in its absence. Structure determination by X-ray crystallography revealed that YcnD folds into a three layer alpha-beta-alpha sandwich strongly resembling the topology of the NADH oxidase superfamily. Similar to homologous bacterial oxidoreductase, YcnD forms homodimers with an extended dimer interface. The biochemical data and the structure are discussed in light of the putative physiological function of YcnD as an oxidoreductase delivering reduced FMN to enzymes that require the reduced cofactor for activity.