Weiqi Pan

Visualizing influenza virus infection in living mice

Preventing and treating influenza virus infection remain a challenge because of incomplete understanding of the host–pathogen interactions, limited therapeutics and lack of a universal vaccine. So far, methods for monitoring the course of infection with influenza virus in real time in living animals are lacking. Here we report the visualization of influenza viral infection in living mice using an engineered replication-competent influenza A virus carrying luciferase reporter gene. After intranasal inoculation, bioluminescence can be detected in the chest and nasopharyngeal passage of living mice. The intensity of bioluminescence in the chest correlates with the dosage of infection and the viral load in the lung. Bioluminescence in the chest of infected mice diminishes on antiviral treatment. This work provides a novel approach that enables real-time study of influenza virus infection and effects of antiviral therapeutics in living animals.

Epidemiology of adenovirus type 5 neutralizing antibodies in healthy people and AIDS patients in Guangzhou, southern China

Recombinant adenovirus serotype 5 (Ad5) viruses have been extensively explored as vectors for vaccination or gene therapy. However, one major obstacle to their clinical application is the high prevalence of preexisting anti-Ad5 immunity resulting from natural infection. It has been reported that there are geographic variations in the prevalence of natural adenovirus infection. In the present study, we investigated the seroprevalence of Ad5 in Guangzhou, southern China by measuring the Ad5 neutralizing antibodies in blood samples collected from several sites. The seroprevalence was 77.34% in the general healthy population. The seroprevalence and antibody titers increased with age, with the older population (41-72 years old) having the highest seropositivity (84.8%) and percentage (54.4%) of high Ad5 neutralizing antibody titers (>1000). The dynamics of Ad5 neutralizing antibodies were stable and persistent over the course of eight months. Furthermore, the seroprevalence of Ad5 in the HIV-infected AIDS patients was investigated and there was no significant difference from the general healthy population. Our survey provides useful insights for the future development of Ad5-based vaccination and gene therapy.

Generation of Replication-Competent Recombinant Influenza A Viruses Carrying a Reporter Gene Harbored in the Neuraminidase Segment

ABSTRACT Replication-competent influenza viruses carrying reporter genes are of great use for basic research, screening of antiviral drugs, and neutralizing of antibodies. In this study, two recombinant influenza A viruses with a neuraminidase (NA) segment harboring enhanced green fluorescent protein (EGFP) in the background of A/PR/8/34 (PR8) were generated. The viral RNA (vRNA)-specific packaging signals for NA were largely retained for efficient packaging. An “autocleave” 2A peptide sequence, which was inserted at the N terminus or the COOH terminus of NA to link with EGFP, enabled NA and EGFP to be expressed monocistronically. Further analysis demonstrated that both viruses, named rPR8-EGFP+NA and rPR8-NA+EGPF, although with some characteristic differences in growth and EGFP expression, could replicate in noncomplementary cells and propagate to large quantities while maintaining genome stability after multiple passages in embryonated eggs. These replication-competent influenza viruses carrying reporter genes are a great addition to the tool set for developing antiviral therapeutics and vaccines and for in vivo studies of viral dissemination and pathogenicity.

Proton channel activity of influenza A virus matrix protein 2 contributes to autophagy arrest

ABSTRACT Influenza A virus infection can arrest autophagy, as evidenced by autophagosome accumulation in infected cells. Here, we report that this autophagosome accumulation can be inhibited by amantadine, an antiviral proton channel inhibitor, in amantadine-sensitive virus infected cells or cells expressing influenza A virus matrix protein 2 (M2). Thus, M2 proton channel activity plays a role in blocking the fusion of autophagosomes with lysosomes, which might be a key mechanism for arresting autophagy.

Rapid Generation of Human-Like Neutralizing Monoclonal Antibodies in Urgent Preparedness for Influenza Pandemics and Virulent Infectious Diseases

Background The outbreaks of emerging infectious diseases caused by pathogens such as SARS coronavirus, H5N1, H1N1, and recently H7N9 influenza viruses, have been associated with significant mortality and morbidity in humans. Neutralizing antibodies from individuals who have recovered from an infection confer therapeutic protection to others infected with the same pathogen. However, survivors may not always be available for providing plasma or for the cloning of monoclonal antibodies (mAbs). Methodology/Principal Findings The genome and the immunoglobulin genes in rhesus macaques and humans are highly homologous; therefore, we investigated whether neutralizing mAbs that are highly homologous to those of humans (human-like) could be generated. Using the H5N1 influenza virus as a model, we first immunized rhesus macaques with recombinant adenoviruses carrying a synthetic gene encoding hemagglutinin (HA). Following screening an antibody phage display library derived from the B cells of immunized monkeys, we cloned selected macaque immunoglobulin heavy chain and light chain variable regions into the human IgG constant region, which generated human-macaque chimeric mAbs exhibiting over 97% homology to human antibodies. Selected mAbs demonstrated potent neutralizing activities against three clades (0, 1, 2) of the H5N1 influenza viruses. The in vivo protection experiments demonstrated that the mAbs effectively protected the mice even when administered up to 3 days after infection with H5N1 influenza virus. In particular, mAb 4E6 demonstrated sub-picomolar binding affinity to HA and superior in vivo protection efficacy without the loss of body weight and obvious lung damage. The analysis of the 4E6 escape mutants demonstrated that the 4E6 antibody bound to a conserved epitope region containing two amino acids on the globular head of HA. Conclusions/Significance Our study demonstrated the generation of neutralizing mAbs for potential application in humans in urgent preparedness against outbreaks of new influenza infections or other virulent infectious diseases.

Improvement of influenza vaccine strain A/Vietnam/1194/2004 (H5N1) growth with the neuraminidase packaging sequence from A/Puerto Rico/8/34.

H5N1 influenza candidate vaccine viruses were developed using the “6+2” approach. The hemagglutinin (HA) and neuraminidase (NA) genes were derived from the popular H5N1 virus and the remaining six internal segments were derived from the A/Puerto Rico/8/34 strain (H1N1, PR8). However, some of these candidate strains have been reported to produce relatively low yields in vaccine manufacture. In this study, we found that the NA vRNA of the A/Vietnam/1194/2004 strain (H5N1, VN1194) was poorly packaged into recombinant viruses with a backbone of PR8 genes, which resulted in the formation of defective virions that did not include the NA vRNA in the genome. Using recombinant DNA techniques, we constructed a chimeric NA gene with the coding region of VN1194 NA flanked by the packaging signal sequence of the PR8 NA gene (41 bp form the 3′ end of the vRNA and 67 bp from the 5′ end). The packaging of the NA vRNA was restored to normal levels in the recombinant viruses containing the chimeric NA gene. Recombinant viruses containing the chimeric NA replicated much better in chicken embryonated eggs than viruses with the wild-type NA from VN1194. These findings suggest a novel strategy to improve in ovo growth of vaccine strains and to increase the number of vaccine doses available to save people if a pandemic were to occur.

Circumventing Antivector Immunity by Using Adenovirus-Infected Blood Cells for Repeated Application of Adenovirus-Vectored Vaccines: Proof of Concept in Rhesus Macaques

ABSTRACT Adenovirus has been extensively exploited as a vector platform for delivering vaccines. However, preexisting antiadenovirus immunity is the major stumbling block for application of adenovirus-vectored vaccines. In this study, we found that freshly isolated peripheral blood mononuclear cells (PBMCs), mostly CD14+ cells, from adenovirus serotype 5 (Ad5)-seropositive primates (humans and rhesus macaques) can be efficiently infected with Ad5 in vitro. On the basis of this observation, a novel strategy based on adenoviral vector-infected PBMC (AVIP) immunization was explored to circumvent antivector immunity. Autologous infusion of Ad5-SIVgag-infected PBMCs elicited a strong Gag-specific cellular immune response but induced weaker Ad5-neutralizing antibody (NAb) in Ad5-seronegative macaques than in macaques intramuscularly injected with Ad5-SIVgag. Moreover, Ad5-seropositive macaques receiving multiple AVIP immunizations with Ad5-SIVenv, Ad5-SIVgag, and Ad5-SIVpol vaccines elicited escalated Env-, Gag-, and Pol-specific immune responses after each immunization that were significantly greater than those in macaques intramuscularly injected with these Ad5-SIV vaccines. After challenged intravenously with a highly pathogenic SIVmac239 virus, macaques receiving AVIP immunization demonstrated a significant reduction in viral load at both the peak time and set-point period compared with macaques without Ad5-SIV vaccines. Our study warranted further research and development of the AVIP immunization as a platform for repeated applications of adenovirus-vectored vaccines.

The mouse Pol I terminator is more efficient than the hepatitis delta virus ribozyme in generating influenza-virus-like RNAs with precise 3′ ends in a plasmid-only-based virus rescue system

Reverse genetics systems for generating recombinant influenza viruses are based on two different mechanisms for obtaining the 3′ end of the viral RNA: one uses the self-cleaving hepatitis delta virus ribozyme (HDVR), and the other uses the murine RNA polymerase I (Pol I) terminator. In this study, we employed EGFP and Renilla luciferase reporter constructs to compare the efficiency of both methods. Our results indicate that the murine Pol I terminator was more efficient than the HDVR, which will be helpful in choosing an influenza virus rescue system, as well as in establishing other RNA virus rescue systems.

Comparative immunogenicity of recombinant adenovirus-vectored vaccines expressing different forms of hemagglutinin (HA) proteins from the H5 serotype of influenza A viruses in mice.

Recent outbreaks of highly pathogenic avian influenza (HPAI) H5N1 viruses in poultry and their subsequent transmission to humans have highlighted an urgent need to develop preventive vaccines in the event of a pandemic. In this paper we constructed recombinant adenovirus (rAd)-vectored influenza vaccines expressing different forms of H5 hemagglutinin (HA) from the A/Vietnam/1194/04 (VN/1194/04) virus, a wild-type HA, a sequence codon-optimized HA and a transmembrane (TM) domain-truncated HA. Compared to the rAd vectors expressing the wild-type HA (rAd-04wtHA) and the TM-truncated form of HA (rAd-04optHA-dTM), the rAd vectored vaccine with the sequence codon-optimized HA (rAd-04optHA) showed a tendency to induce much higher hemagglutinin inhibition (HI) antibody titers in mice immunized with a prime-boost vaccine. Furthermore, administration of the rAd-04optHA vaccine to mice could elicit cross-reactive immune responses against the antigenically distinct HK/482/97 virus. Additionally, we constructed another vector containing the codon-optimized HA of the A/Hong Kong/482/97 (HK/482/97) virus. Administration of a bivalent immunization formulation including the rAd-04optHA and rAd-97optHA vaccines to mice induced a stronger immune response against HK/482/97 virus than the monovalent formulation. Taken together, these findings may have some implications for the development of rAd-vectored vaccines in the event of the pandemic spread of HPAI.

Induction of neutralizing antibodies to influenza A virus H7N9 by inactivated whole virus in mice and nonhuman primates

We evaluated the immunogenicity of hemagglutinin (HA) in the context of inactivated H7N9/AH/1/13-PR8 whole-virion. At 4weeks after immunization with 15μg HA, mice produced hemagglutination inhibition (HI) titers of 1:192 and neutralizing antibodies of 1:317. Aluminum hydroxide (alum), or a booster immunization, or both increased HI to 1:768, 1:384, 1:896 and neutralizing antibodies to 1:1868, 1:2302, 1:10,000, respectively. Macaques generated HI of 1:190 or 1:360 and virus neutralizing titers of 1:280 or 1:658 at 3weeks after immunization with HA alone or with alum. Sera from immunized mice and macaques protected mice from infection of A/Anhui/1/2013 (H7N9), suggesting an H7N9 vaccine is immunologically feasible.