Aleksandr S. Lipatov

In vitro evolution of H5N1 avian influenza virus toward human-type receptor specificity.

Acquisition of α2-6 sialoside receptor specificity by α2-3 specific highly-pathogenic avian influenza viruses (H5N1) is thought to be a prerequisite for efficient transmission in humans. By in vitro selection for binding α2-6 sialosides, we identified four variant viruses with amino acid substitutions in the hemagglutinin (S227N, D187G, E190G, and Q196R) that revealed modestly increased α2-6 and minimally decreased α2-3 binding by glycan array analysis. However, a mutant virus combining Q196R with mutations from previous pandemic viruses (Q226L and G228S) revealed predominantly α2-6 binding. Unlike the wild type H5N1, this mutant virus was transmitted by direct contact in the ferret model although not by airborne respiratory droplets. However, a reassortant virus with the mutant hemagglutinin, a human N2 neuraminidase and internal genes from an H5N1 virus was partially transmitted via respiratory droplets. The complex changes required for airborne transmissibility in ferrets suggest that extensive evolution is needed for H5N1 transmissibility in humans.

Induction of Long-Term Protective Immune Responses by Influenza H5N1 Virus-Like Particles

Background Recurrent outbreaks of highly pathogenic H5N1 avian influenza virus pose a threat of eventually causing a pandemic. Early vaccination of the population would be the single most effective measure for the control of an emerging influenza pandemic. Methodology/Principal Findings Influenza virus-like particles (VLPs) produced in insect cell-culture substrates do not depend on the availability of fertile eggs for vaccine manufacturing. We produced VLPs containing influenza A/Viet Nam1203/04 (H5N1) hemagglutinin, neuraminidase, and matrix proteins, and investigated their preclinical immunogenicity and protective efficacy. Mice immunized intranasally with H5N1 VLPs developed high levels of H5N1 specific antibodies and were 100% protected against a high dose of homologous H5N1 virus infection at 30 weeks after immunization. Protection is likely to be correlated with humoral and cellular immunologic memory at systemic and mucosal sites as evidenced by rapid anamnestic responses to re-stimulation with viral antigen in vivo and in vitro. Conclusions/Significance These results provide support for clinical evaluation of H5N1 VLP vaccination as a public health intervention to mitigate a possible pandemic of H5N1 influenza.

Protective immunity against H5N1 influenza virus by a single dose vaccination with virus-like particles

We generated influenza virus-like particles (VLPs) containing the wild type (WT) H5 hemagglutinin (HA) from A/Viet Nam/1203/04 virus or a mutant H5 HA with a deletion of the multibasic cleavage motif. VLPs containing mutant H5 HA were found to be as immunogenic as VLPs containing WT HA. A single intramuscular vaccination with either type of H5 VLPs provided complete protection against lethal challenge. In contrast, the recombinant H5 HA vaccine was less immunogenic and vaccination even with a 5 fold higher dose did not induce protective immunity. VLP vaccines were superior to the recombinant HA in inducing T helper type 1 immune responses, hemagglutination inhibition titers, and antibody secreting cells, which significantly contribute to inducing protective immunity after a single dose vaccination. This study provides insights into the potential mechanisms of improved immunogenicity by H5 VLP vaccines as an approach to improve the protective efficacy against potential pandemic viruses.

The development of vaccine viruses against pandemic A(H1N1) influenza

Wild type human influenza viruses do not usually grow well in embryonated hens eggs, the substrate of choice for the production of inactivated influenza vaccine, and vaccine viruses need to be developed specifically for this purpose. In the event of a pandemic of influenza, vaccine viruses need to be created with utmost speed. At the onset of the current A(H1N1) pandemic in April 2009, a network of laboratories began a race against time to develop suitable candidate vaccine viruses. Two approaches were followed, the classical reassortment approach and the more recent reverse genetics approach. This report describes the development and the characteristics of current pandemic H1N1 candidate vaccine viruses.

Microneedle Delivery of H5N1 Influenza Virus-Like Particles to the Skin Induces Long-Lasting B- and T-Cell Responses in Mice

ABSTRACT A simple method suitable for self-administration of vaccine would improve mass immunization, particularly during a pandemic outbreak. Influenza virus-like particles (VLPs) have been suggested as promising vaccine candidates against potentially pandemic influenza viruses, as they confer long-lasting immunity but are not infectious. We investigated the immunogenicity and protective efficacy of influenza H5 VLPs containing the hemagglutinin (HA) of A/Vietnam/1203/04 (H5N1) virus delivered into the skin of mice using metal microneedle patches and also studied the response of Langerhans cells in a human skin model. Prime-boost microneedle vaccinations with H5 VLPs elicited higher levels of virus-specific IgG1 and IgG2a antibodies, virus-specific antibody-secreting cells, and cytokine-producing cells up to 8 months after vaccination compared to the same antigen delivered intramuscularly. Both prime-boost microneedle and intramuscular vaccinations with H5 VLPs induced similar hemagglutination inhibition titers and conferred 100% protection against lethal challenge with the wild-type A/Vietnam/1203/04 virus 16 weeks after vaccination. Microneedle delivery of influenza VLPs to viable human skin using microneedles induced the movement of CD207+ Langerhans cells toward the basement membrane. Microneedle vaccination in the skin with H5 VLPs represents a promising approach for a self-administered vaccine against viruses with pandemic potential.

Increased immunogenicity of avian influenza DNA vaccine delivered to the skin using a microneedle patch

Effective public health responses to an influenza pandemic require an effective vaccine that can be manufactured and administered to large populations in the shortest possible time. In this study, we evaluated a method for vaccination against avian influenza virus that uses a DNA vaccine for rapid manufacturing and delivered by a microneedle skin patch for simplified administration and increased immunogenicity. We prepared patches containing 700-μm long microneedles coated with an avian H5 influenza hemagglutinin DNA vaccine from A/Viet Nam/1203/04 influenza virus. The coating DNA dose increased with DNA concentration in the coating solution and the number of dip-coating cycles. Coated DNA was released into the skin tissue by dissolution within minutes. Vaccination of mice using microneedles induced higher levels of antibody responses and hemagglutination inhibition titers, and improved protection against lethal infection with avian influenza as compared to conventional intramuscular delivery of the same dose of the DNA vaccine. Additional analysis showed that the microneedle coating solution containing carboxymethylcellulose and a surfactant may have negatively affected the immunogenicity of the DNA vaccine. Overall, this study shows that DNA vaccine delivery by microneedles can be a promising approach for improved vaccination to mitigate an influenza pandemic.

The Virulence of 1997 H5N1 Influenza Viruses in the Mouse Model Is Increased by Correcting a Defect in Their NS1 Proteins

ABSTRACT The NS1 protein of human influenza A viruses binds the 30-kDa subunit of the cleavage and polyadenylation specificity factor (CPSF30), a protein required for 3′ end processing of cellular pre-mRNAs, thereby inhibiting production of beta interferon (IFN-β) mRNA. The NS1 proteins of pathogenic 1997 H5N1 viruses contain the CPSF30-binding site but lack the consensus amino acids at positions 103 and 106, F and M, respectively, that are required for the stabilization of CPSF30 binding, resulting in nonoptimal CPSF30 binding in infected cells. Here we have demonstrated that strengthening CPSF30 binding, by changing positions 103 and 106 in the 1997 H5N1 NS1 protein to the consensus amino acids, results in a remarkable 300-fold increase in the lethality of the virus in mice. Unexpectedly, this increase in virulence is not associated with increased lung pathology but rather is characterized by faster systemic spread of the virus, particularly to the brain, where increased replication and severe pathology occur. This increased spread is associated with increased cytokine and chemokine levels in extrapulmonary tissues. We conclude that strengthening CPSF30 binding by the NS1 protein of 1997 H5N1 viruses enhances virulence in mice by increasing the systemic spread of the virus from the lungs, particularly to the brain.

Virus-Like Particle Vaccine Containing Hemagglutinin Confers Protection against 2009 H1N1 Pandemic Influenza

ABSTRACT Immunization of the world population before an influenza pandemic such as the 2009 H1N1 virus spreads globally is not possible with current vaccine production platforms. New influenza vaccine technologies, such as virus-like-particles (VLPs), offer a promising alternative. Here, we tested the immunogenicity and protective efficacy of a VLP vaccine containing hemagglutinin (HA) and M1 from the 2009 pandemic H1N1 influenza virus (H1N1pdm) in ferrets and compared intramuscular (i.m.) and intranasal (i.n.) routes of immunization. Vaccination of ferrets with VLPs containing the M1 and HA proteins from A/California/04/2009 (H1N1pdm) induced high antibody titers and conferred significant protection against virus challenge. VLP-vaccinated animals lost less weight, shed less virus in nasal washes, and had markedly lower virus titers in all organs tested than naïve controls. A single dose of VLPs, either i.m. or i.n., induced higher levels of antibody than did two doses of commercial split vaccine. Ferrets vaccinated with split vaccine were incompletely protected against challenge; these animals had lower virus titers in olfactory bulbs, tonsils, and intestines, but lost weight and shed virus in nasal washes to a similar extent as naïve controls. Challenge with heterologous A/Brisbane/59/07 (H1N1) virus revealed that the VLPs conferred minimal cross-protection to heterologous infection, as revealed by the lack of reduction in nasal wash and lung virus titers and slightly higher weight loss relative to controls. In summary, these experiments demonstrate the strong immunogenicity and protective efficacy of VLPs compared to the split vaccine and show that i.n. vaccination with VLPs has the potential for highly efficacious vaccination against influenza.

Influenza vaccination accelerates recovery of ferrets from lymphopenia.

Ferrets are a useful animal model for human influenza virus infections, since they closely mimic the pathogenesis of influenza viruses observed in humans. However, a lack of reagents, especially for flow cytometry of immune cell subsets, has limited research in this model. Here we use a panel of primarily species cross-reactive antibodies to identify ferret T cells, cytotoxic T lymphocytes (CTL), B cells, and granulocytes in peripheral blood. Following infection with seasonal H3N2 or H1N1pdm09 influenza viruses, these cell types showed rapid and dramatic changes in frequency, even though clinically the infections were mild. The loss of B cells and CD4 and CD8 T cells, and the increase in neutrophils, were especially marked 1–2 days after infection, when about 90% of CD8+ T cells disappeared from the peripheral blood. The different virus strains led to different kinetics of leukocyte subset alterations. Vaccination with homologous vaccine reduced clinical symptoms slightly, but led to a much more rapid return to normal leukocyte parameters. Assessment of clinical symptoms may underestimate the effectiveness of influenza vaccine in restoring homeostasis.

Flexible Label-Free Quantitative Assay for Antibodies to Influenza Virus Hemagglutinins

ABSTRACT During the initial pandemic influenza H1N1 virus outbreak, assays such as hemagglutination inhibition and microneutralization provided important information on the relative protection afforded by the populations cross-reactivity from prior infections and immunizations with seasonal vaccines. However, these assays continue to be limited in that they are difficult to automate for high throughput, such as in pandemic situations, as well as to standardize between labs. Thus, new technologies are being sought to improve standardization, reliability, and throughput by using chemically defined reagents rather than whole cells and virions. We now report the use of a cell-free and label-free flu antibody biosensor assay (f-AbBA) for influenza research and diagnostics that utilizes recombinant hemagglutinin (HA) in conjunction with label-free biolayer interferometry technology to measure biomolecular interactions between the HA and specific anti-HA antibodies or sialylated ligands. We evaluated f-AbBA to determine anti-HA antibody binding activity in serum or plasma to assess vaccine-induced humoral responses. This assay can reveal the impact of antigenic difference on antibody binding to HA and also measure binding to different subtypes of HA. We also show that the biosensor assay can measure the ability of HA to bind a model sialylated receptor-like ligand. f-AbBA could be used in global surveillance laboratories since preliminary tests on desiccated HA probes showed no loss of activity after >2 months in storage at room temperature, indicating that the same reagent lots could be used in different laboratories to minimize interlaboratory assay fluctuation. Future development of such reagents and similar technologies may offer a robust platform for future influenza surveillance activities.