Lethal H5N1 Influenza Research Articles

Influenza virus immune imprinting dictates the clinical outcomes in ferrets challenged with highly pathogenic avian influenza virus H5N1.

Zoonotic transmission of H5N1 highly pathogenic avian influenza virus (HPAIV) into the human population is an increasing global threat. The recent 2022 HPAIV outbreak significantly highlighted this possibility, increasing concern in the general population. The clinical outcomes of H5N1 influenza virus exposure can be determined by an individual's primary influenza virus infection (imprinting) or vaccination status. Immunological imprinting with Group 1 - (H1N1, H2N2, and H2N3) increases survival rates following H5N1 viral infection compared to Group 2 - (H3N2) imprinted individuals. Vaccination against H5N1 influenza viruses can offer protection to at-risk populations; however, stockpiled inactivated H5N1 influenza vaccines are not readily available to the public. We hypothesize that the immunological response to vaccination and subsequent clinical outcome following H5N1 influenza virus infection is correlated with the immunological imprinting status of an individual. To test this hypothesis, our lab established a ferret pre-immune model of disease. Naïve ferrets were intranasally inoculated with seasonal influenza viruses and allowed to recover for 84 days prior to H5N1 virus infection. Ferrets imprinted following H1N1 and H2N3 virus infections were completely protected against lethal H5N1 influenza virus challenge (100% survival), with few to no clinical symptoms. In comparison, H3N2 influenza virus-imprinted ferrets had severe clinical symptoms, delayed disease progression, and a sublethal phenotype (40% mortality). Consecutive infections with H1N1 influenza viruses followed by an H3N2 influenza virus infection did not abrogate the immune protection induced by the original H1N1 influenza virus infection. In addition, ferrets consecutively infected with H1N1 and H2N3 viruses had no clinical symptoms or weight loss. H3N2 pre-immune ferrets were vaccinated with a broadly reactive H5 HA-based or H1 NA-based vaccine (Hu-CO 2). These ferrets were protected against H5N1 influenza virus challenge, whereas ferrets vaccinated with the H1N1 wild-type CA/09 rHA vaccine had similar phenotypes as non-vaccinated H3N2-imprinted ferrets with 40% survival. Overall, Group 2 imprinted ferrets, which were vaccinated with heterologous Group 1 HA vaccines, had redirected immune responses to Group 1 influenza viral antigens and rescued a sublethal phenotype to complete protection.

Infiltration of inflammatory macrophages and neutrophils and widespread pyroptosis in lung drive influenza lethality in nonhuman primates.

Severe influenza kills tens of thousands of individuals each year, yet the mechanisms driving lethality in humans are poorly understood. Here we used a unique translational model of lethal H5N1 influenza in cynomolgus macaques that utilizes inhalation of small-particle virus aerosols to define mechanisms driving lethal disease. RNA sequencing of lung tissue revealed an intense interferon response within two days of infection that resulted in widespread expression of interferon-stimulated genes, including inflammatory cytokines and chemokines. Macaques with lethal disease had rapid and profound loss of alveolar macrophages (AMs) and infiltration of activated CCR2+ CX3CR1+ interstitial macrophages (IMs) and neutrophils into lungs. Parallel changes of AMs and neutrophils in bronchoalveolar lavage (BAL) correlated with virus load when compared to macaques with mild influenza. Both AMs and IMs in lethal influenza were M1-type inflammatory macrophages which expressed neutrophil chemotactic factors, while neutrophils expressed genes associated with activation and generation of neutrophil extracellular traps (NETs). NETs were prominent in lung and were found in alveolar spaces as well as lung parenchyma. Genes associated with pyroptosis but not apoptosis were increased in lung, and activated inflammatory caspases, IL-1β and cleaved gasdermin D (GSDMD) were present in bronchoalveolar lavage fluid and lung homogenates. Cleaved GSDMD was expressed by lung macrophages and alveolar epithelial cells which were present in large numbers in alveolar spaces, consistent with loss of epithelial integrity. Cleaved GSDMD colocalized with viral NP-expressing cells in alveoli, reflecting pyroptosis of infected cells. These novel findings reveal that a potent interferon and inflammatory cascade in lung associated with infiltration of inflammatory macrophages and neutrophils, elaboration of NETs and cell death by pyroptosis mediates lethal H5N1 influenza in nonhuman primates, and by extension humans. These innate pathways represent promising therapeutic targets to prevent severe influenza and potentially other primary viral pneumonias in humans.

Mini-hemagglutinin vaccination induces cross-reactive antibodies in pre-exposed NHP that protect mice against lethal influenza challenge

Seasonal vaccines are currently the most effective countermeasure against influenza. However, seasonal vaccines are only effective against strains closely related to the influenza strains contained in the vaccine. Recently a new hemagglutinin (HA) stem-based antigen, the so-called “mini-HA”, has been shown to induce a cross-protective immune response in influenza-naive mice and non-human primates (NHP). However, prior exposure to influenza can have a profound effect on the immune response to subsequent influenza infection and the protective efficacy of vaccination. Here we show that mini-HA, compared to a trivalent influenza vaccine (TIV), elicits a broadened influenza-specific humoral immune response in NHP previously exposed to influenza. Serum transfer experiments showed that antibodies induced by both mini-HA and seasonal vaccine protected mice against lethal challenge with a H1N1 influenza strain heterologous to the H1 HA included in the TIV. However, antibodies elicited by mini-HA showed an additional benefit of protecting mice against lethal heterosubtypic H5N1 influenza challenge, associated with H5 HA-specific functional antibodies.

Furan-carboxamide derivatives as novel inhibitors of lethal H5N1 influenza A viruses

The simple scaffold furan-carboxamide derivatives were firstly identified as novel inhibitors of lethal H5N1 influenza A virus.

Protective Efficacy of an H5N1 Inactivated Vaccine Against Challenge with Lethal H5N1, H5N2, H5N6, and H5N8 Influenza Viruses in Chickens.

The Goose/Guangdong-lineage H5 viruses have evolved into diverse clades and subclades based on their hemagglutinin (HA) gene during their circulation in wild birds and poultry. Since late 2013, the clade 2.3.4.4 viruses have become widespread in poultry and wild bird populations around the world. Different subtypes of the clade 2.3.4.4 H5 viruses, including H5N1, H5N2, H5N6, and H5N8, have caused vast disease outbreaks in poultry in Asia, Europe, and North America. In this study, we developed a new H5N1 inactivated vaccine by using a seed virus (designated as Re-8) that contains the HA and NA genes from a clade 2.3.4.4 virus, A/chicken/Guizhou/4/13(H5N1) (CK/GZ/4/13), and its six internal genes from the high-growth A/Puerto Rico/8/1934 (H1N1) virus. We evaluated the protective efficacy of this vaccine in chickens challenged with one H5N1 clade 2.3.2.1b virus and six different subtypes of clade 2.3.4.4 viruses, including H5N1, H5N2, H5N6, and H5N8 strains. In the clade 2.3.2.1b virus DK/GX/S1017/13-challenged groups, half of the vaccinated chickens shed virus through the oropharynx and two birds (20%) died during the observation period. All of the control chickens shed viruses and died within 6 days of infection with challenge virus. All of the vaccinated chickens remained healthy following challenge with the six clade 2.3.4.4 viruses, and virus shedding was not detected from any of these birds; however, all of the control birds shed viruses and died within 4 days of challenge with the clade 2.3.4.4 viruses. Our results indicate that the Re-8 vaccine provides protection against different subtypes of clade 2.3.4.4 H5 viruses.

ID: 35: The dimer interfaces of B7-2 and CD28 receptors control their engagement and signaling and are effective therapeutic targets for lethal cytokine storm

We created peptides that protect broadly against severe, life-threatening infections and reveal a novel and critical regulatory role for costimulatory receptor homodimer interfaces in inflammatory T-cell hyperactivation. Our finding is that the interaction between B7-2 and CD28, primary costimulatory receptors that are expressed constitutively, can be controlled through their dimer interfaces and presents a novel, broad-spectrum host-oriented target for harmful inflammatory cytokine responses (“cytokine storm”). Excessive induction of inflammatory cytokines by Gram-positive bacterial superantigens, we show, strictly requires their direct binding into the homodimer interfaces of CD28 and its coligand, B7-2. By binding the dimer interface of both receptors, superantigens potently enhance B7-2/CD28 engagement and signaling, to induce a cytokine storm. Short peptide mimetics of the B7-2 or CD28 dimer interface bind diverse superantigens, prevent binding of superantigen to cell-surface B7-2 or CD28, attenuate superantigen-induced overexpression of IFN- γ , TNF- α and IL2 in human PBMC, and are effective antagonists in vivo, protecting mice from lethal superantigen challenge. B7-2 and CD28 dimer interfaces thus serve as novel therapeutic targets for superantigen intoxication. Surprisingly, when used in pathologies unrelated to superantigens, peptide mimetics of either the CD28 or B7-2 dimer interface potently attenuate the inflammatory cytokine storm, by directly inhibiting B7-2/CD28 engagement. In human PBMC, these peptides inhibit induction of inflammatory cytokines by lipopolysaccharide, a hallmark of Gram-negative infection, or by monoclonal antibodies that signal through CD28. Such host-oriented peptides protect mice from lethal avian H5N1 and pandemic H1N1 influenza virus infection while strongly reducing inflammatory cytokine levels.

Hemagglutinin-stem nanoparticles generate heterosubtypic influenza protection.

The antibody response to influenza is primarily focused on the head region of the hemagglutinin (HA) glycoprotein, which in turn undergoes antigenic drift, thus necessitating annual updates of influenza vaccines. In contrast, the immunogenically subdominant stem region of HA is highly conserved and recognized by antibodies capable of binding multiple HA subtypes. Here we report the structure-based development of an H1 HA stem-only immunogen that confers heterosubtypic protection in mice and ferrets. Six iterative cycles of structure-based design (Gen1-Gen6) yielded successive H1 HA stabilized-stem (HA-SS) immunogens that lack the immunodominant head domain. Antigenic characterization, determination of two HA-SS crystal structures in complex with stem-specific monoclonal antibodies and cryo-electron microscopy analysis of HA-SS on ferritin nanoparticles (H1-SS-np) confirmed the preservation of key structural elements. Vaccination of mice and ferrets with H1-SS-np elicited broadly cross-reactive antibodies that completely protected mice and partially protected ferrets against lethal heterosubtypic H5N1 influenza virus challenge despite the absence of detectable H5N1 neutralizing activity in vitro. Passive transfer of immunoglobulin from H1-SS-np-immunized mice to naive mice conferred protection against H5N1 challenge, indicating that vaccine-elicited HA stem-specific antibodies can protect against diverse group 1 influenza strains.

MTOR regulates protection against lethal H5N1 influenza infection by modulating the antibody response (VAC2P.926)

Abstract Highly pathogenic H5N1 avian influenza viruses pose a continuing global threat. Current vaccines will not protect against novel pandemic viruses. Creating “universal” vaccines has been unsuccessful because the immunological mechanisms promoting heterosubtypic immunity are incompletely defined. We show that rapamycin, an immunosuppressive drug that inhibits mTOR, promotes cross-strain protection against a lethal H5N1 infection when administered during H3N2 virus immunization. Rapamycin reduced germinal center formation and inhibited B cell class-switching, yielding a unique repertoire of antibodies that mediated heterosubtypic protection. Our data establish a requirement for mTORC1 in B cell class-switching and demonstrate that rapamycin skews the antibody response away from high affinity variant epitopes, targeting more conserved elements of hemagglutinin. Our current data indicate that a primary intranasal infection with FluMist enhances heterosubtypic immunity even more, which can be further enhanced with rapamycin. In this model the CD8 T cells contribute to enhanced protection providing us a model to study the synergy between cross-reactive antibodies and CD8 T cells. These findings have intriguing implications for influenza vaccine design.

MTOR regulates protection against a lethal H5N1 influenza infection by modulating the antibody response (P6184)

Abstract Highly pathogenic H5N1 avian influenza A viruses pose a serious threat for global catastrophe. Due to frequent mutations in the influenza genome, current vaccination protocols promote only strain-specific responses, and therefore fail to provide protection against novel pandemic strains. Antibodies that protect against multiple subtypes of influenza have been identified, however, these antibodies are extremely rare, and the immunological mechanisms that lead to their development are not clear. We show here that rapamycin, an immunosuppressive drug that inhibits mTOR, promoted cross-strain protection against lethal H5N1 infection when administered during H3N2 vaccination. Surprisingly, this protection did not require memory CD8 T cells, but rather required CD4 T cells and B cells, suggesting the protection was antibody-mediated. In support of this, serum from rapamycin-treated mice immunized with a H3N2 strain protected naïve mice from a lethal H5N1 infection. Our data further reveal that rapamycin reduced germinal center formation and B cell class-switching, yielding a unique repertoire of antibodies that was more protective against a lethal, heterosubtypic influenza infection. These data not only reveal a requirement for mTOR in B cell class-switching, but also indicate that a broader antibody repertoire, which provides better protection against multiple strains of influenza, can be achieved by reducing germinal center formation during the primary infection.

Neuraminidase-Inhibiting Antibody Is a Correlate of Cross-Protection against Lethal H5N1 Influenza Virus in Ferrets Immunized with Seasonal Influenza Vaccine

In preparing for the threat of a pandemic of avian H5N1 influenza virus, we need to consider the significant delay (4 to 6 months) necessary to produce a strain-matched vaccine. As some degree of cross-reactivity between seasonal influenza vaccines and H5N1 virus has been reported, this was further explored in the ferret model to determine the targets of protective immunity. Ferrets were vaccinated with two intramuscular inoculations of trivalent inactivated split influenza vaccine or subcomponent vaccines, with and without adjuvant, and later challenged with a lethal dose of A/Vietnam/1203/2004 (H5N1) influenza virus. We confirmed that vaccination with seasonal influenza vaccine afforded partial protection against lethal H5N1 challenge and showed that use of either AlPO(4) or Iscomatrix adjuvant with the vaccine resulted in complete protection against disease and death. The protection was due exclusively to the H1N1 vaccine component, and although the hemagglutinin contributed to protection, the dominant protective response was targeted toward the neuraminidase (NA) and correlated with sialic acid cleavage-inhibiting antibody titers. Purified heterologous NA formulated with Iscomatrix adjuvant was also protective. These results suggest that adjuvanted seasonal trivalent vaccine could be used as an interim measure to decrease morbidity and mortality from H5N1 prior to the availability of a specific vaccine. The data also highlight that an inducer of cross-protective immunity is the NA, a protein whose levels are not normally monitored in vaccines and whose capacity to induce immunity in recipients is not normally assessed.

MDA5 Can Be Exploited as Efficacious Genetic Adjuvant for DNA Vaccination against Lethal H5N1 Influenza Virus Infection in Chickens

Chickens lack the retinoic acid-inducible gene I (RIG-I) and sense avian influenza virus (AIV) infections by means of the melanoma differentiation-associated gene 5 product (chMDA5). Plasmid-driven expression of the N-terminal half of chMDA5 containing the caspase activation and recruitment domains [chMDA5(1-483)] triggers interferon-β responses in chicken cells. We hypothesized that mimicking virus infection by chMDA5(1-483) expression may enhance vaccine-induced adaptive immunity. In order to test this, the potential genetic adjuvant properties of chMDA5(1-483) were evaluated in vivo in combination with a suboptimal quantity of a plasmid DNA vaccine expressing haemagglutinin (HA) of H5N1 AIV. Co-administration of the HA plasmid with plasmid DNA for chMDA5(1-483) expression resulted in approximately 10-fold higher HA-specific antibody responses than injection of the HA plasmid mixed with empty vector DNA as control. Accordingly, compared with HA DNA vaccination alone, the chMDA5(1-483)-adjuvanted HA DNA vaccine mediated enhanced protection against a lethal H5N1 challenge infection in chickens, with reduced clinical signs and cloacal virus shedding. These data demonstrate that innate immune activation by expression of signaling domains of RIG-I-like receptors can be exploited to enhance vaccine efficacy.

Lethal H5N1 influenza viruses are not resistant to interferon action in human, simian, porcine or chicken cells

Lethal H5N1 influenza viruses are not resistant to interferon action in human, simian, porcine or chicken cells

Changing Realities in Emerging Zoonoses

In the 3 years since this International Conference on Emerging Zoonoses was last held, there have been significant changes in both the spread of numerous zoonoses and the manner in which zoonoses, both new and old, are perceived by scientists, governments and the public. Although the term ‘foreign animal/human diseases’ is still widely used, there is near unanimous awareness that such diseases are transboundary diseases, in which an outbreak in any country can spread rapidly throughout the world. Therefore, the recent outbreaks of Hendra virus in Australia and Nipah virus in Bangladesh constitute significant disease threats not only in those countries but in any country. Furthermore, the sporadic occurrences of the highly lethal H5N1 influenza virus and the existence of more transmissible human and avian influenza viruses of different subtypes threaten both animals and people with the dangers of new reassortants. The danger of such an unwanted event has not lessened, whatever the amount of publicity about influenza. It is crucial to understand that we cannot predict which emerging zoonosis in which country will suddenly become a disease of epidemic proportions. Rift Valley Fever, African swine fever and dengue fever are perhaps the three emerging diseases most in need of immediate vaccine development. However, Ebola-Marburg viral diseases, Hendra and Nipah viral diseases, and SARS (Severe Acute Respiratory Syndrome)—the first pandemic of the 21st century—are just as likely to become the next focus of international concern. Meanwhile, malaria continues to kill many people in Africa and Asia with impunity, despite encouraging advances in vaccine development and treatment. In the midst of such a complex and changing disease environment, those gathered for this Sixth International Conference on Emerging Zoonoses sought to share their own work and to listen to the ideas and hopes of other scientists. The flippant phrase about the need ‘to network’ to advance one's career is a gross travesty of the purpose of any international conference, for it is only by meeting and sharing and listening that we can hope to improve the quality and relevance of our research. None of us have all the answers, but hopefully some of us have a few of the answers. Rather, it is not so much ‘answers’ that we seek but sensible preparation grounded in alert anticipation of the options for where and when the next significant disease outbreaks might occur. No doubt there will be multiple unexpected disease events before we next gather in 2014 in Berlin. There is a high probability that the zoonotic disease that concerns us most at the next conference will be some newly emerging disease, such as the recently discovered Schmallenberg virus. However, there is one reality that will not change. Veterinary medicine, human medicine and environmental awareness are the three foundations on which One Health is based and the only way that we can successfully confront changing realities in emerging and re-emerging zoonoses.

Pentamers Not Found in the Universal Proteome Can Enhance Antigen Specific Immune Responses and Adjuvant Vaccines

Certain short peptides do not occur in humans and are rare or non-existent in the universal proteome. Antigens that contain rare amino acid sequences are in general highly immunogenic and may activate different arms of the immune system. We first generated a list of rare, semi-common, and common 5-mer peptides using bioinformatics tools to analyze the UniProtKB database. Experimental observations indicated that rare and semi-common 5-mers generated stronger cellular responses in comparison with common-occurring sequences. We hypothesized that the biological process responsible for this enhanced immunogenicity could be used to positively modulate immune responses with potential application for vaccine development. Initially, twelve rare 5-mers, 9-mers, and 13-mers were incorporated in frame at the end of an H5N1 hemagglutinin (HA) antigen and expressed from a DNA vaccine. The presence of some 5-mer peptides induced improved immune responses. Adding one 5-mer peptide exogenously also offered improved clinical outcome and/or survival against a lethal H5N1 or H1N1 influenza virus challenge in BALB/c mice and ferrets, respectively. Interestingly, enhanced anti-HBsAg antibody production by up to 25-fold in combination with a commercial Hepatitis B vaccine (Engerix-B, GSK) was also observed in BALB/c mice. Mechanistically, NK cell activation and dependency was observed with enhancing peptides ex vivo and in NK-depleted mice. Overall, the data suggest that rare or non-existent oligopeptides can be developed as immunomodulators and supports the further evaluation of some 5-mer peptides as potential vaccine adjuvants.

Protection against a lethal H5N1 influenza challenge by intranasal immunization with virus-like particles containing 2009 pandemic H1N1 neuraminidase in mice

Highly pathogenic H5N1 influenza shares the same neuraminidase (NA) subtype with the 2009 pandemic (H1N1pdm09), and cross-reactive NA immunity might protect against or mitigate lethal H5N1 infection. In this study, mice were either infected with a sublethal dose of H1N1pdm09 or were vaccinated and boosted with virus-like particles (VLP) consisting of the NA and matrix proteins, standardized by NA activity and administered intranasally, and were then challenged with a lethal dose of HPAI H5N1 virus. Mice previously infected with H1N1pdm09 survived H5N1 challenge with no detectable virus or respiratory tract pathology on day 4. Mice immunized with H5N1 or H1N1pdm09 NA VLPs were also fully protected from death, with a 100-fold and 10-fold reduction in infectious virus, respectively, and reduced pathology in the lungs. Human influenza vaccines that elicit not only HA, but also NA immunity may provide enhanced protection against the emergence of seasonal and pandemic viruses.

Proteomic analysis of the lungs of mice infected with different pathotypes of H5N1 avian influenza viruses

The virulence of influenza virus is determined by viral and host factors. Data on the genetic basis of the virulence of H5N1 influenza viruses have increased over the past decade; however, the contributions of host factors to the outcomes of H5N1 infection remain largely unknown. Here, we tested two chicken H5N1 viruses in mice and found that A/chicken/VN1214/2007 was nonlethal in mice and only replicated in the lung, whereas A/chicken/VN1180/2006 was highly lethal and replicated systemically in mice. To investigate the host response against these two different virus infections, we performed proteomic analysis by using 2D DIGE on the lung tissues of mice collected on days 1 and 3 postinoculation with different viruses or PBS as a control. Thirty-nine differentially expressed (DE) proteins related to "immune and stimulus response," "macromolecular biosynthesis and metabolism," and "cellular component and cytoskeleton" were identified in the virus-inoculated groups. Moreover, 13 DE proteins were identified between the two virus-inoculated groups, implying that these proteins may play important roles in the different outcomes of infection with these two viruses. Our data provide important information regarding the host response to mild and lethal H5N1 influenza virus infection.

Antisense oligonucleotides targeting the RNA binding region of the NP gene inhibit replication of highly pathogenic avian influenza virus H5N1

The H5N1 avian influenza virus (AIV) causes widespread infections in bird and human respiratory tracts, and vaccines and drug therapy are limited in their effectiveness. Recent studies of AIV structures have been published and provide new targets for designing antiviral drugs such as antisense oligonucleotides (AS ODNs), which effectively inhibit gene replication. In this study, we designed and synthesized three AS ODNs (NP267, NP628, NP749) that were specific for the RNA binding region of nucleoprotein (NP) based on AIV structure. Results showed that all three AS ODNs could inhibit viral replication in MDCK cells. The NP628 showed the best antiviral effect of all through viral titers, quantitative RT-PCR and indirect immunofluorescence (IFA) assays. In addition, the liposome mediated NP628 could partially protect the mice from a lethal H5N1 influenza virus challenge. Moreover, the NP628 group had a lower viral titer and lung index in the infected mice when compared with the viral control. Our results showed that AS ODN targeting of the AIV NP gene could potently inhibit AIV H5N1 reproduction, thus, formulating a candidate for an emergent therapeutic drug for the pathogenic H5N1 influenza virus infection.

Antisense oligonucleotide inhibits avian influenza virus H5N1 replication by single chain antibody delivery system

H5N1 avian influenza virus (AIV) causes widespread infections in poultry and wild birds, and has the potential to emerge as a pandemic threat to human. Antisense oligonucleotides (AS ODNs) are highly effective at inhibiting gene replication. Antibody-mediated delivery is a novel approach to target specific cells and tissues. In this study, we designed and synthesized three AS ODNs (PA4, PA492 and PA1203) specific for conserved region of AIV PA protein, and all the three AS ODNs could inhibit viral replication. The PA492 ODN showed the best antiviral effect by viral titers and quantitative RT-PCR in MDCK cells. The fusion protein scFv-tP was constructed as a single chain variable fragment (scFv) against AIV hemaglutinin antigen with a truncated protamine (tP). The results showed that scFv-tP fusion improved the antiviral effectiveness of PA492 in MDCK cells as measured by viral titers, quantitative RT-PCR and indirect immunofluorescence (IFA) assays. In addition, scFv-tP-delivered PA492 was also found to partially protect mice from lethal H5N1 influenza virus challenge. Using scFv-tP delivery, fluorescein isothiocyanate labeled-PA492 was found to be significantly localized in the lungs, compared to liposome-delivered PA492. Moreover, the fusion protein mediated PA492 had a lower lung index and viral titers in the infected mice as compared with the liposome method. These results provided a potential method for using anti-HA fusion protein for the targeted delivery of AS ODNs against AIV H5N1.

Vaccination with hemagglutinin produced in Trichoplusia ni larvae protects chickens against lethal H5N1 challenge

Highly pathogenic avian influenza (HPAI) H5N1 viruses in birds and coincident infections in humans have created demand for a cost-effective vaccine to prevent a pandemic of the disease. We report here that Trichoplusia ni ( T. ni) larvae can act as a cost-effective bioreactor to produce recombinant HA5 (rH5HA) proteins as an effective vaccine for chickens. Western blot analysis revealed that the 70 kDa rH5HA protein and partially cleaved products (40 kDa rH5HA1 and 28 kDa rH5HA2) were generated in T. ni larvae infected with recombinant baculovirus carrying the H5HA gene. We demonstrated that a single intramuscular injection of homogenates of T. ni larvae containing rH5HA proteins into chickens could induce the production of antibodies with hemagglutination inhibition (HI) capability and stimulate neutralization activity against a lethal H5N1 influenza virus challenge. Furthermore, each vAc-HA5-infected T. ni larva can vaccinate approximately 20–36 chickens and induce with HI titer values of approximately 5.5 log 2 after 7 weeks implied that this single-shot protocol should be economical and provide adequate protection. These data suggest that the baculovirus-larvae recombinant protein expression system could be a cost-effective platform for vaccine production.

Distinct pathological signatures after lethal avian H5N1 and swine H1N1 influenza infections suggest variable pathogenesis

Distinct pathological signatures after lethal avian H5N1 and swine H1N1 influenza infections suggest variable pathogenesis