Amorsolo L. Suguitan

Live, Attenuated Influenza A H5N1 Candidate Vaccines Provide Broad Cross-Protection in Mice and Ferrets

Background Recent outbreaks of highly pathogenic influenza A H5N1 viruses in humans and avian species that began in Asia and have spread to other continents underscore an urgent need to develop vaccines that would protect the human population in the event of a pandemic. Methods and Findings Live, attenuated candidate vaccines possessing genes encoding a modified H5 hemagglutinin (HA) and a wild-type (wt) N1 neuraminidase from influenza A H5N1 viruses isolated in Hong Kong and Vietnam in 1997, 2003, and 2004, and remaining gene segments derived from the cold-adapted (ca) influenza A vaccine donor strain, influenza A/Ann Arbor/6/60 ca (H2N2), were generated by reverse genetics. The H5N1 ca vaccine viruses required trypsin for efficient growth in vitro, as predicted by the modification engineered in the gene encoding the HA, and possessed the temperature-sensitive and attenuation phenotypes specified by the internal protein genes of the ca vaccine donor strain. More importantly, the candidate vaccines were immunogenic in mice. Four weeks after receiving a single dose of 106 50% tissue culture infectious doses of intranasally administered vaccines, mice were fully protected from lethality following challenge with homologous and antigenically distinct heterologous wt H5N1 viruses from different genetic sublineages (clades 1, 2, and 3) that were isolated in Asia between 1997 and 2005. Four weeks after receiving two doses of the vaccines, mice and ferrets were fully protected against pulmonary replication of homologous and heterologous wt H5N1 viruses. Conclusions The promising findings in these preclinical studies of safety, immunogenicity, and efficacy of the H5N1 ca vaccines against antigenically diverse H5N1 vaccines provide support for their careful evaluation in Phase 1 clinical trials in humans.

Prophylactic and therapeutic efficacy of human monoclonal antibodies against H5N1 influenza.

Background New prophylactic and therapeutic strategies to combat human infections with highly pathogenic avian influenza (HPAI) H5N1 viruses are needed. We generated neutralizing anti-H5N1 human monoclonal antibodies (mAbs) and tested their efficacy for prophylaxis and therapy in a murine model of infection. Methods and Findings Using Epstein-Barr virus we immortalized memory B cells from Vietnamese adults who had recovered from infections with HPAI H5N1 viruses. Supernatants from B cell lines were screened in a virus neutralization assay. B cell lines secreting neutralizing antibodies were cloned and the mAbs purified. The cross-reactivity of these antibodies for different strains of H5N1 was tested in vitro by neutralization assays, and their prophylactic and therapeutic efficacy in vivo was tested in mice. In vitro, mAbs FLA3.14 and FLD20.19 neutralized both Clade I and Clade II H5N1 viruses, whilst FLA5.10 and FLD21.140 neutralized Clade I viruses only. In vivo, FLA3.14 and FLA5.10 conferred protection from lethality in mice challenged with A/Vietnam/1203/04 (H5N1) in a dose-dependent manner. mAb prophylaxis provided a statistically significant reduction in pulmonary virus titer, reduced associated inflammation in the lungs, and restricted extrapulmonary dissemination of the virus. Therapeutic doses of FLA3.14, FLA5.10, FLD20.19, and FLD21.140 provided robust protection from lethality at least up to 72 h postinfection with A/Vietnam/1203/04 (H5N1). mAbs FLA3.14, FLD21.140 and FLD20.19, but not FLA5.10, were also therapeutically active in vivo against the Clade II virus A/Indonesia/5/2005 (H5N1). Conclusions These studies provide proof of concept that fully human mAbs with neutralizing activity can be rapidly generated from the peripheral blood of convalescent patients and that these mAbs are effective for the prevention and treatment of H5N1 infection in a mouse model. A panel of neutralizing, cross-reactive mAbs might be useful for prophylaxis or adjunctive treatment of human cases of H5N1 influenza.

Glycosylation at 158N of the Hemagglutinin Protein and Receptor Binding Specificity Synergistically Affect the Antigenicity and Immunogenicity of a Live Attenuated H5N1 A/Vietnam/1203/2004 Vaccine Virus in Ferrets

ABSTRACT A live attenuated influenza A/Vietnam/1203/2004 (H5N1) vaccine virus (VN04 ca) has receptor binding specificity to α2,3-linked sialosides (α2,3SAL), and a single dose induces a minimal serum antibody response in mice and ferrets. In contrast, A/Hong Kong/213/2003 (H5N1) vaccine virus (HK03 ca) binds to both α2,6SAL and α2,3SAL and generates a stronger serum antibody response in animals. Among the 9 amino acids that differed between the two H5 HA1 proteins, several HK03-specific residues enabled the VN04 ca virus to bind to both α2,3SAL and α2,6SAL receptors, but only the removal of the 158N glycosylation, together with an S227N change, resulted in more-efficient viral replication in the upper respiratory tract of ferrets and an increased serum antibody response. However, the antibody response was HK03 strain specific and did not significantly cross-neutralize VN04 virus. A second approach was taken to adapt the H5N1 VN04 ca virus in MDCK cells to select HA variants with larger plaque morphology. Although a number of large-plaque-size HA variants with amino acid changes in the HA receptor binding region were identified, none of these mutations affected virus receptor binding preference and immunogenicity. In addition, the known receptor binding site changes, Q226L and G228S, were introduced into the HA protein of the VN04 ca virus. Only in conjunction with the removal of the 158N glycosylation did the virus replicate efficiently in the upper respiratory tract of ferrets and became more immunogenic, yet the response was also HK03 specific. Thus, the mask of the antigenic epitopes by 158N glycosylation at the HA globular head and its α2,3SAL binding preference of VN04 ca virus affect virus antigenicity and replication in the host, resulting in a lower antibody response.

Generation of Live Attenuated Novel Influenza Virus A/California/7/09 (H1N1) Vaccines with High Yield in Embryonated Chicken Eggs

ABSTRACT Several live attenuated influenza virus A/California/7/09 (H1N1) (CA09) candidate vaccine variants that possess the hemagglutinin (HA) and neuraminidase (NA) gene segments from the CA09 virus and six internal protein gene segments from the cold-adapted influenza virus A/Ann Arbor/6/60 (H2N2) virus were generated by reverse genetics. The reassortant viruses replicated relatively poorly in embryonated chicken eggs. To improve virus growth in eggs, reassortants expressing the HA and NA of CA09 were passaged in MDCK cells and variants exhibiting large-plaque morphology were isolated. These variants replicated at levels approximately 10-fold higher than the rate of replication of the parental strains in embryonated chicken eggs. Sequence analysis indicated that single amino acid changes at positions 119, 153, 154, and 186 were responsible for the improved growth properties in MDCK cells and eggs. In addition, the introduction of a mutation at residue 155 that was previously shown to enhance the replication of a 1976 swine influenza virus also significantly improved the replication of the CA09 virus in eggs. Each variant was further evaluated for receptor binding preference, antigenicity, attenuation phenotype, and immunogenicity. Mutations at residues 153, 154, and 155 drastically reduced viral antigenicity, which made these mutants unsuitable as vaccine candidates. However, changes at residues 119 and 186 did not affect virus antigenicity or immunogenicity, justifying their inclusion in live attenuated vaccine candidates to protect against the currently circulating 2009 swine origin H1N1 viruses.

Evaluation of two live attenuated cold-adapted H5N1 influenza virus vaccines in healthy adults

BACKGROUND Development of live attenuated influenza vaccines (LAIV) against avian viruses with pandemic potential is an important public health strategy. METHODS AND FINDINGS We performed open-label trials to evaluate the safety, infectivity, and immunogenicity of H5N1 VN 2004 AA ca and H5N1 HK 2003 AA ca. Each of these vaccines contains a modified H5 hemagglutinin and unmodified N1 neuraminidase from the respective wild-type (wt) parent virus and the six internal protein gene segments of the A/Ann Arbor/6/60 cold-adapted (ca) master donor virus. The H5N1 VN 2004 AA ca vaccine virus was evaluated at dosages of 10(6.7) TCID(50) and 10(7.5) TCID(50), and the H5N1 HK 2003 AA ca vaccine was evaluated at a dosage of 10(7.5) TCID(50). Two doses were administered intranasally to healthy adults in isolation at 4-8 week intervals. Vaccine safety was assessed through daily examinations and infectivity was assessed by viral culture and by realtime reverse transcription-polymerase chain reaction testing of nasal wash (NW) specimens. Immunogenicity was assessed by measuring hemagglutination-inhibition (HI) antibodies, neutralizing antibodies, and IgG or IgA antibodies to recombinant (r)H5 VN 2004 hemagglutinin (HA) in serum or NW. Fifty-nine participants were enrolled: 21 received 10(6.7) TCID(50) and 21 received 10(7.5) TCID(50) of H5N1 VN 2004 AA ca and 17 received H5N1 HK 2003 AA ca. Shedding of vaccine virus was minimal, as were HI and neutralizing antibody responses. Fifty-two percent of recipients of 10(7.5) TCID(50) of H5N1 VN 2004 AA ca developed a serum IgA response to rH5 VN 2004 HA. CONCLUSIONS The live attenuated H5N1 VN 2004 and HK 2003 AA ca vaccines bearing avian H5 HA antigens were very restricted in replication and were more attenuated than seasonal LAIV bearing human H1, H3 or B HA antigens. The H5N1 AA ca LAIV elicited serum ELISA antibody but not HI or neutralizing antibody responses in healthy adults. (ClinicalTrials.gov Identifiers: NCT00347672 and NCT00488046).

Immunization of Primates with a Newcastle Disease Virus-Vectored Vaccine via the Respiratory Tract Induces a High Titer of Serum Neutralizing Antibodies against Highly Pathogenic Avian Influenza Virus

ABSTRACT The ongoing outbreak of highly pathogenic avian influenza virus (HPAIV) in birds, the incidence of transmission to humans with a resulting high mortality rate, and the possibility of a human pandemic warrant the development of effective human vaccines against HPAIV. We developed an experimental live-attenuated vaccine for direct inoculation of the respiratory tract based on recombinant avian Newcastle disease virus (NDV) expressing the hemagglutinin (HA) glycoprotein of H5N1 HPAIV (NDV-HA). Expression of the HPAIV HA gene slightly reduced NDV virulence, as evidenced by the increased mean embryo death time and reduced replication in chickens. NDV-HA was administered to African green monkeys in two doses of 2 × 107 infectious units each with a 28-day interval to evaluate the systemic and local antibody responses specific to H5N1 HPAIV. The virus was shed only at low titers from the monkeys, indicative of safety. Two doses of NDV-HA induced a high titer of H5N1 HPAIV-neutralizing serum antibodies in all of the immunized monkeys. Moreover, a substantial mucosal immunoglobulin A response was induced in the respiratory tract after one and two doses. The titers of neutralizing antibodies achieved in this study suggest that the vaccine would be likely to prevent mortality and reduce morbidity caused by the H5N1 HPAIV. In addition, induction of a local immune response in the respiratory tract is an important advantage that is likely to reduce or prevent transmission of the virus during an outbreak or a pandemic. This vaccine is a candidate for clinical evaluation in humans.

The Multibasic Cleavage Site of the Hemagglutinin of Highly Pathogenic A/Vietnam/1203/2004 (H5N1) Avian Influenza Virus Acts as a Virulence Factor in a Host-Specific Manner in Mammals

ABSTRACT Highly pathogenic avian influenza (HPAI) viruses of the H5 and H7 subtypes typically possess multiple basic amino acids around the cleavage site (MBS) of their hemagglutinin (HA) protein, a recognized virulence motif in poultry. To determine the importance of the H5 HA MBS as a virulence factor in mammals, recombinant wild-type HPAI A/Vietnam/1203/2004 (H5N1) viruses that possessed (H5N1) or lacked (ΔH5N1) the H5 HA MBS were generated and evaluated for their virulence in BALB/c mice, ferrets, and African green monkeys (AGMs) (Chlorocebus aethiops). The presence of the H5 HA MBS was associated with lethality, significantly higher virus titers in the respiratory tract, virus dissemination to extrapulmonary organs, lymphopenia, significantly elevated levels of proinflammatory cytokines and chemokines, and inflammation in the lungs of mice and ferrets. In AGMs, neither H5N1 nor ΔH5N1 virus was lethal and neither caused clinical symptoms. The H5 HA MBS was associated with mild enhancement of replication and delayed virus clearance. Thus, the contribution of H5 HA MBS to the virulence of the HPAI H5N1 virus varies among mammalian hosts and is most significant in mice and ferrets and less remarkable in nonhuman primates.

Potent Vesicular Stomatitis Virus-Based Avian Influenza Vaccines Provide Long-Term Sterilizing Immunity against Heterologous Challenge

ABSTRACT The emergence in 1997 and continuance today of a highly lethal H5N1 avian influenza virus (AIV) causing human disease has raised concern about an impending pandemic and the need for a vaccine to prepare for such an occurrence. We previously generated an efficacious vesicular stomatitis virus (VSV)-based AIV vaccine expressing H5 hemagglutinin (HA) from the fifth genomic position of VSV (J. A. Schwartz et al., Virology 366:166-173, 2007). Here we have generated and characterized VSV-based vaccines that express the A/Hong Kong/156/1997 (clade 0) H5 HA from the first position of the VSV genome. These vectors induce broadly cross-neutralizing antibodies against homologous and heterologous H5N1 viruses of different clades in mice. The vaccines provide complete protection against morbidity and mortality after heterologous challenge with clade 0 and clade 1 strains in animals even 1 year after vaccination. Postchallenge pulmonary virus loads show that these vectors provide sterilizing immunity. Therefore, VSV-based AIV vaccines are potent, broadly cross-protective pandemic vaccine candidates.

The influence of the multi-basic cleavage site of the H5 hemagglutinin on the attenuation, immunogenicity and efficacy of a live attenuated influenza A H5N1 cold-adapted vaccine virus.

A recombinant live attenuated influenza virus DeltaH5N1 vaccine with a modified hemagglutinin (HA) and intact neuraminidase genes from A/Vietnam/1203/04 (H5N1) and six remaining genome segments from A/Ann Arbor/6/60 (H2N2) cold-adapted (AA ca) virus was previously shown to be attenuated in chickens, mice and ferrets. Evaluation of the recombinant H5N1 viruses in mice indicated that three independent factors contributed to the attenuation of the DeltaH5N1 vaccine: the attenuating mutations specified by the AA ca loci had the greatest influence, followed by the deletion of the H5 HA multi-basic cleavage site (MBS), and the constellation effects of the AA genes acting in concert with the H5N1 glycoproteins. Restoring the MBS in the H5 HA of the vaccine virus improved its immunogenicity and efficacy, likely as a consequence of increased virus replication, indicating that removal of the MBS had a deleterious effect on the immunogenicity and efficacy of the DeltaH5N1 vaccine in mice.

Vesicular Stomatitis Virus-Based H5N1 Avian Influenza Vaccines Induce Potent Cross-Clade Neutralizing Antibodies in Rhesus Macaques

ABSTRACT We analyzed the ability of a vaccine vector based on vesicular stomatitis virus (VSV) to induce a neutralizing antibody (NAb) response to avian influenza viruses (AIVs) in rhesus macaques. Animals vaccinated with vectors expressing either strain A/Hong Kong/156/1997 or strain A/Vietnam/1203/2004 H5 hemagglutinin (HA) were able to generate robust NAb responses. The ability of the vectors to induce NAbs against homologous and heterologous AIVs after a single dose was dependent upon the HA antigen incorporated into the VSV vaccine. The vectors expressing strain A/Vietnam/1203/2004 H5 HA were superior to those expressing strain A/Hong Kong/156/1997 HA at inducing cross-clade NAbs.