H3 Stalk-Based Chimeric Hemagglutinin Influenza Virus Constructs Protect Mice from H7N9 Challenge

The recent outbreak of H7N9 influenza virus infections in humans in China has raised concerns about the pandemic potential of this strain. Here, we test the efficacy of H3 stalk-based chimeric hemagglutinin universal influenza virus vaccine constructs to protect against H7N9 challenge in mice. Chimeric hemagglutinin constructs protected from viral challenge in the context of different administration routes as well as with a generic oil-in-water adjuvant similar to formulations licensed for use in humans.     

Monoclonal Antibodies against the Fusion Peptide of Hemagglutinin Protect Mice from Lethal Influenza A Virus H5N1 Infection

The HA2 glycopolypeptide (gp) is highly conserved in all influenza A virus strains, and it is known to play a major role in the fusion of the virus with the endosomal membrane in host cells during the course of viral infection. Vaccines and therapeutics targeting this HA2 gp could induce efficient broad-spectrum immunity against influenza A virus infections. So far, there have been no studies on the possible therapeutic effects of monoclonal antibodies (MAbs), specifically against the fusion peptide of hemagglutinin (HA), upon lethal infections with highly pathogenic avian influenza (HPAI) H5N1 virus. We have identified MAb 1C9, which binds to GLFGAIAGF, a part of the fusion peptide of the HA2 gp. We evaluated the efficacy of MAb 1C9 as a therapy for influenza A virus infections. This MAb, which inhibited cell fusion in vitro when administered passively, protected 100% of mice from challenge with five 50% mouse lethal doses of HPAI H5N1 influenza A viruses from two different clades. Furthermore, it caused earlier clearance of the virus from the lung. The influenza virus load was assessed in lung samples from mice challenged after pretreatment with MAb 1C9 (24 h prior to challenge) and from mice receiving early treatment (24 h after challenge). The study shows that MAb 1C9, which is specific to the antigenically conserved fusion peptide of HA2, can contribute to the cross-clade protection of mice infected with H5N1 virus and mediate more effective recovery from infection.Highly pathogenic avian influenza (HPAI) virus H5N1 strains are currently causing major morbidity and mortality in poultry populations across Asia, Europe, and Africa and have caused 385 confirmed human infections, with a fatality rate of 63.11% (37, 39). Preventive and therapeutic measures against circulating H5N1 strains have received a lot of interest and effort globally to prevent another pandemic outbreak. Influenza A virus poses a challenge because it rapidly alters its appearance to the immune system by antigenic drift (mutating) and antigenic shift (exchanging its components) (5). The current strategies to combat influenza include vaccination and antiviral drug treatment, with vaccination being the preferred option. The annual influenza vaccine aims to stimulate the generation of anti-hemagglutinin (anti-HA) neutralizing antibodies, which confer protection against homologous strains. Current vaccines have met with various degrees of success (31). The facts that these strategies target the highly variable HA determinant and that predicting the major HA types that pose the next epidemic threat is difficult are significant limitations to the current antiviral strategy. In the absence of an effective vaccine, therapy is the mainstay of control of influenza virus infection.Therefore, therapeutic measures against influenza will play a major role in case a pandemic arises due to H5N1 strains. Currently licensed antiviral drugs include the M2 ion-channel inhibitors (rimantidine and amantidine) and the neuraminidase inhibitors (oseltamivir and zanamivir). The H5N1 viruses are known to be resistant to the M2 ion-channel inhibitors (2, 3). Newer strains of H5N1 viruses are being isolated which are also resistant to the neuraminidase inhibitors (oseltamivir and zanamivir) (5, 17). The neuraminidase inhibitors also require high doses and prolonged treatment (5, 40), increasing the likelihood of unwanted side effects. Hence, alternative strategies for treatment of influenza are warranted.Recently, passive immunotherapy using monoclonal antibodies (MAbs) has been viewed as a viable option for treatment (26). The HA gene is the most variable gene of the influenza virus and also the most promising target for generating antibodies. It is synthesized as a precursor polypeptide, HA0, which is posttranslationally cleaved to two polypeptides, HA1 and HA2, linked by a disulfide bond. MAbs against the HA1 glycopolypeptide (gp) are known to neutralize the infectivity of the virus and hence provide good protection against infection (12). However, they are less efficient against heterologous or mutant strains, which are continuously arising due to antigenic shift and, to an extent, drift. Recent strategies for alternative therapy explore the more conserved epitopes of the influenza virus antigens (18, 33), which not only have the potential to stimulate a protective immune response but are also conserved among different subtypes, so as to offer protection against a broader range of viruses.The HA2 polypeptide represents a highly conserved region of HA across influenza A virus strains. The HA2 gp is responsible for the fusion of the virus and the host endosomal membrane during the entry of the virus into the cell (16). Previously, anti-HA MAbs that lacked HA inhibition activity were studied and were found to reduce the infectivity of non-H5 influenza virus subtypes by inhibition of fusion during viral replication (14). They are known to block fusion of the virus to the cell membrane at the postbinding and prefusion stage, thereby inhibiting viral replication. Furthermore, in vivo studies show that anti-HA2 MAbs that exhibit fusion inhibition activity contribute to protection and recovery from H3N2 influenza A virus infection (8). It is interesting that although the HA2 gp is generally conserved, the fusion peptide represents the most conserved region of the HA protein. So far, there have been no studies on the possible therapeutic effects of MAbs, specifically against the fusion peptide of HA, on lethal HPAI H5N1 infections.Previous studies have suggested that HA2 could contain a potential epitope responsible for the induction of antibody-mediated protective immunity (9). In the present study, a panel of MAbs against HA2 gp was characterized for their respective epitopes by epitope mapping. The therapeutic and prophylactic efficacies of these MAbs were evaluated in mice challenged with HPAI H5N1 virus infection.     

Recombinant Parainfluenza Virus 5 Expressing Hemagglutinin of Influenza A Virus H5N1 Protected Mice against Lethal Highly Pathogenic Avian Influenza Virus H5N1 Challenge

A safe and effective vaccine is the best way to prevent large-scale highly pathogenic avian influenza virus (HPAI) H5N1 outbreaks in the human population. The current FDA-approved H5N1 vaccine has serious limitations. A more efficacious H5N1 vaccine is urgently needed. Parainfluenza virus 5 (PIV5), a paramyxovirus, is not known to cause any illness in humans. PIV5 is an attractive vaccine vector. In our studies, a single dose of a live recombinant PIV5 expressing a hemagglutinin (HA) gene of H5N1 (rPIV5-H5) from the H5N1 subtype provided sterilizing immunity against lethal doses of HPAI H5N1 infection in mice. Furthermore, we have examined the effect of insertion of H5N1 HA at different locations within the PIV5 genome on the efficacy of a PIV5-based vaccine. Interestingly, insertion of H5N1 HA between the leader sequence, the de facto promoter of PIV5, and the first viral gene, nucleoprotein (NP), did not lead to a viable virus. Insertion of H5N1 HA between NP and the next gene, V/phosphorprotein (V/P), led to a virus that was defective in growth. We have found that insertion of H5N1 HA at the junction between the small hydrophobic (SH) gene and the hemagglutinin-neuraminidase (HN) gene gave the best immunity against HPAI H5N1 challenge: a dose as low as 1,000 PFU was sufficient to protect against lethal HPAI H5N1 challenge in mice. The work suggests that recombinant PIV5 expressing H5N1 HA has great potential as an HPAI H5N1 vaccine.     

Amino Acid Substitutions Improve the Immunogenicity of H7N7HA Protein and Protect Mice against Lethal H7N7 Viral Challenge

Avian influenza A H7N7/NL/219/03 virus creates a serious pandemic threat to human health because it can transmit directly from domestic poultry to humans and from human to human. Our previous vaccine study reported that mice when immunized intranasally (i.n) with live Bac-HA were protected from lethal H7N7/NL/219/03 challenge, whereas incomplete protection was obtained when administered subcutaneously (s.c) due to the fact that H7N7 is a poor inducer of neutralizing antibodies. Interestingly, our recent vaccine studies reported that mice when vaccinated subcutaneously with Bac-HA (H7N9) was protected against both H7N9 (A/Sh2/2013) and H7N7 virus challenge. HA1 region of both H7N7 and H7N9 viruses are differ at 15 amino acid positions. Among those, we selected three amino acid positions (T143, T198 and I211) in HA1 region of H7N7. These amino acids are located within or near the receptor binding site. Following the selection, we substituted the amino acid at these three positions with amino acids found on H7N9HA wild-type. In this study, we evaluate the impact of amino acid substitutions in the H7N7 HA-protein on the immunogenicity. We generated six mutant constructs from wild-type influenza H7N7HA cDNA by site directed mutagenesis, and individually expressed mutant HA protein on the surface of baculovirus (Bac-HA_m) and compared their protective efficacy of the vaccines with Bac-H7N7HA wild-type (Bac-HA) by lethal H7N7 viral challenge in a mouse model. We found that mice immunized subcutaneously with Bac-HA_m constructs T143A or T198A-I211V or I211V-T143A serum showed significantly higher hemagglutination inhibition and neutralization titer against H7N7 and H7N9 viruses when compared to Bac-HA vaccinated mice groups. We also observed low level of lung viral titer, negligible weight loss and complete protection against lethal H7N7 viral challenge. Our results indicated that amino acid substitution at position 143 or 211 improve immunogenicity of H7N7HA vaccine against H7N7/NL/219/03 virus.     

Liposome-Antigen-Nucleic Acid Complexes Protect Mice from Lethal Challenge with Western and Eastern Equine Encephalitis Viruses

Alphaviruses are mosquito-borne viruses that cause significant disease in animals and humans. Western equine encephalitis virus (WEEV) and eastern equine encephalitis virus (EEEV), two New World alphaviruses, can cause fatal encephalitis, and EEEV is a select agent of concern in biodefense. However, we have no antiviral therapies against alphaviral disease, and current vaccine strategies target only a single alphavirus species. In an effort to develop new tools for a broader response to outbreaks, we designed and tested a novel alphavirus vaccine comprised of cationic lipid nucleic acid complexes (CLNCs) and the ectodomain of WEEV E1 protein (E1ecto). Interestingly, we found that the CLNC component, alone, had therapeutic efficacy, as it increased survival of CD-1 mice following lethal WEEV infection. Immunization with the CLNC-WEEV E1ecto mixture (lipid-antigen-nucleic acid complexes [LANACs]) using a prime-boost regimen provided 100% protection in mice challenged with WEEV subcutaneously, intranasally, or via mosquito. Mice immunized with LANACs mounted a strong humoral immune response but did not produce neutralizing antibodies. Passive transfer of serum from LANAC E1ecto-immunized mice to nonimmune CD-1 mice conferred protection against WEEV challenge, indicating that antibody is sufficient for protection. In addition, the LANAC E1ecto immunization protocol significantly increased survival of mice following intranasal or subcutaneous challenge with EEEV. In summary, our LANAC formulation has therapeutic potential and is an effective vaccine strategy that offers protection against two distinct species of alphavirus irrespective of the route of infection. We discuss plausible mechanisms as well the potential utility of our LANAC formulation as a pan-alphavirus vaccine.     

Protection of Mice against Lethal Challenge with 2009 H1N1 Influenza A Virus by 1918-Like and Classical Swine H1N1 Based Vaccines

The recent 2009 pandemic H1N1 virus infection in humans has resulted in nearly 5,000 deaths worldwide. Early epidemiological findings indicated a low level of infection in the older population (>65 years) with the pandemic virus, and a greater susceptibility in people younger than 35 years of age, a phenomenon correlated with the presence of cross-reactive immunity in the older population. It is unclear what virus(es) might be responsible for this apparent cross-protection against the 2009 pandemic H1N1 virus. We describe a mouse lethal challenge model for the 2009 pandemic H1N1 strain, used together with a panel of inactivated H1N1 virus vaccines and hemagglutinin (HA) monoclonal antibodies to dissect the possible humoral antigenic determinants of pre-existing immunity against this virus in the human population. By hemagglutinination inhibition (HI) assays and vaccination/challenge studies, we demonstrate that the 2009 pandemic H1N1 virus is antigenically similar to human H1N1 viruses that circulated from 1918–1943 and to classical swine H1N1 viruses. Antibodies elicited against 1918-like or classical swine H1N1 vaccines completely protect C57B/6 mice from lethal challenge with the influenza A/Netherlands/602/2009 virus isolate. In contrast, contemporary H1N1 vaccines afforded only partial protection. Passive immunization with cross-reactive monoclonal antibodies (mAbs) raised against either 1918 or A/California/04/2009 HA proteins offered full protection from death. Analysis of mAb antibody escape mutants, generated by selection of 2009 H1N1 virus with these mAbs, indicate that antigenic site Sa is one of the conserved cross-protective epitopes. Our findings in mice agree with serological data showing high prevalence of 2009 H1N1 cross-reactive antibodies only in the older population, indicating that prior infection with 1918-like viruses or vaccination against the 1976 swine H1N1 virus in the USA are likely to provide protection against the 2009 pandemic H1N1 virus. This data provides a mechanistic basis for the protection seen in the older population, and emphasizes a rationale for including vaccination of the younger, naïve population. Our results also support the notion that pigs can act as an animal reservoir where influenza virus HAs become antigenically frozen for long periods of time, facilitating the generation of human pandemic viruses.     

Properly Folded Bacterially Expressed H1N1 Hemagglutinin Globular Head and Ectodomain Vaccines Protect Ferrets against H1N1 Pandemic Influenza Virus

Background

In the face of impending influenza pandemic, a rapid vaccine production and mass vaccination is the most effective approach to prevent the large scale mortality and morbidity that was associated with the 1918 “Spanish Flu”. The traditional process of influenza vaccine production in eggs is time consuming and may not meet the demands of rapid global vaccination required to curtail influenza pandemic.

Methodology/Principal Findings

Recombinant technology can be used to express the hemagglutinin (HA) of the emerging new influenza strain in a variety of systems including mammalian, insect, and bacterial cells. In this study, two forms of HA proteins derived from the currently circulating novel H1N1 A/California/07/2009 virus, HA1 (1–330) and HA (1–480), were expressed and purified from E. coli under controlled redox refolding conditions that favoured proper protein folding. However, only the recombinant HA1 (1–330) protein formed oligomers, including functional trimers that bound receptor and caused agglutination of human red blood cells. These proteins were used to vaccinate ferrets prior to challenge with the A/California/07/2009 virus. Both proteins induced neutralizing antibodies, and reduced viral loads in nasal washes. However, the HA1 (1–330) protein that had higher content of multimeric forms provided better protection from fever and weight loss at a lower vaccine dose compared with HA (1–480). Protein yield for the HA1 (1–330) ranged around 40 mg/Liter, while the HA (1–480) yield was 0.4–0.8 mg/Liter.

Conclusions/Significance

This is the first study that describes production in bacterial system of properly folded functional globular HA1 domain trimers, lacking the HA2 transmembrane protein, that elicit potent neutralizing antibody responses following vaccination and protect ferrets from in vivo challenge. The combination of bacterial expression system with established quality control methods could provide a mechanism for rapid large scale production of influenza vaccines in the face of influenza pandemic threat.     

Immunization with a Live Attenuated H7N9 Influenza Vaccine Protects Mice against Lethal Challenge

The emergence of severe cases of human influenza A (H7N9) viral infection in China in the spring of 2003 resulted in a global effort to rapidly develop an effective candidate vaccine. In this study, a cold-adapted (ca), live attenuated monovalent reassortant influenza H7N9 virus (Ah01/AA ca) was generated using reverse genetics that contained hemagglutinin (HA) and neuraminidase (NA) genes from a 2013 pandemic A H7N9 isolate, A/Anhui/01/2013 virus (Ah01/H7N9); the remaining six backbone genes derived from the cold-adapted influenza H2N2 A/Ann Arbor/6/60 virus (AA virus). Ah01/AA ca virus exhibited temperature sensitivity (ts), ca, and attenuation (att) phenotypes. Intranasal immunization of female BALB/c mice with Ah01/AA ca twice at a 2-week interval induced robust humoral, mucosal, and cell-mediated immune responses in a dose-dependent manner. Furthermore, the candidate Ah01/AA ca virus was immunogenic and offered partial or complete protection of mice against a lethal challenge by the live 2013 influenza A H7N9 (A/Anhui/01/2013). Protection was demonstrated by the inhibition of viral replication and the attenuation of histopathological changes in the challenged mouse lung. Taken together, these data support the further evaluation of this Ah01/AA ca candidate vaccine in primates.     

Stepwise Engineering of Heterodimeric Single Domain Camelid VHH Antibodies That Passively Protect Mice from Ricin Toxin

In an effort to engineer countermeasures for the category B toxin ricin, we produced and characterized a collection of epitopic tagged, heavy chain-only antibody V_H domains (V_HHs) specific for the ricin enzymatic (RTA) and binding (RTB) subunits. Among the 20 unique ricin-specific V_HHs we identified, six had toxin-neutralizing activity: five specific for RTA and one specific for RTB. Three neutralizing RTA-specific V_HHs were each linked via a short peptide spacer to the sole neutralizing anti-RTB V_HH to create V_HH “heterodimers.” As compared with equimolar concentrations of their respective monovalent monomers, all three V_HH heterodimers had higher affinities for ricin and, in the case of heterodimer D10/B7, a 6-fold increase in in vitro toxin-neutralizing activity. When passively administered to mice at a 4:1 heterodimer:toxin ratio, D10/B7 conferred 100% survival in response to a 10 × LD₅₀ ricin challenge, whereas a 2:1 heterodimer:toxin ratio conferred 20% survival. However, complete survival was achievable when the low dose of D10/B7 was combined with an IgG1 anti-epitopic tag monoclonal antibody, possibly because decorating the toxin with up to four IgGs promoted serum clearance. The two additional ricin-specific heterodimers, when tested in vivo, provided equal or greater passive protection than D10/B7, thereby warranting further investigation of all three heterodimers as possible therapeutics.     

Isolation of Single-Stranded DNA Aptamers That Distinguish Influenza Virus Hemagglutinin Subtype H1 from H5

Surface protein hemagglutinin (HA) mediates the binding of influenza virus to host cell receptors containing sialic acid, facilitating the entry of the virus into host cells. Therefore, the HA protein is regarded as a suitable target for the development of influenza virus detection devices. In this study, we isolated single-stranded DNA (ssDNA) aptamers binding to the HA1 subunit of subtype H1 (H1-HA1), but not to the HA1 subunit of subtype H5 (H5-HA1), using a counter-systematic evolution of ligands by exponential enrichment (counter-SELEX) procedure. Enzyme-linked immunosorbent assay and surface plasmon resonance studies showed that the selected aptamers bind tightly to H1-HA1 with dissociation constants in the nanomolar range. Western blot analysis demonstrated that the aptamers were binding to H1-HA1 in a concentration-dependent manner, yet were not binding to H5-HA1. Interestingly, the selected aptamers contained G-rich sequences in the central random nucleotides region. Further biophysical analysis showed that the G-rich sequences formed a G-quadruplex structure, which is a distinctive structure compared to the starting ssDNA library. Using flow cytometry analysis, we found that the aptamers did not bind to the receptor-binding site of H1-HA1. These results indicate that the selected aptamers that distinguish H1-HA1 from H5-HA1 can be developed as unique probes for the detection of the H1 subtype of influenza virus.     

Newcastle Disease Virus-Vectored H7 and H5 Live Vaccines Protect Chickens from Challenge with H7N9 or H5N1 Avian Influenza Viruses

Sporadic human infections by a novel H7N9 virus occurred over a large geographic region in China. In this study, we show that Newcastle disease virus (NDV)-vectored H7 (NDV-H7) and NDV-H5 vaccines are able to induce antibodies with high hemagglutination inhibition (HI) titers and completely protect chickens from challenge with the novel H7N9 or highly pathogenic H5N1 viruses, respectively. Notably, a baculovirus-expressed H7 protein failed to protect chickens from H7N9 virus infection.     

Second Generation Inactivated Eastern Equine Encephalitis Virus Vaccine Candidates Protect Mice against a Lethal Aerosol Challenge

Currently, there are no FDA-licensed vaccines or therapeutics for eastern equine encephalitis virus (EEEV) for human use. We recently developed several methods to inactivate CVEV1219, a chimeric live-attenuated eastern equine encephalitis virus (EEEV). Dosage and schedule studies were conducted to evaluate the immunogenicity and protective efficacy of three potential second-generation inactivated EEEV (iEEEV) vaccine candidates in mice: formalin-inactivated CVEV1219 (fCVEV1219), INA-inactivated CVEV1219 (iCVEV1219) and gamma-irradiated CVEV1219 (gCVEV1219). Both fCVEV1219 and gCVEV1219 provided partial to complete protection against an aerosol challenge when administered by different routes and schedules at various doses, while iCVEV1219 was unable to provide substantial protection against an aerosol challenge by any route, dose, or schedule tested. When evaluating antibody responses, neutralizing antibody, not virus specific IgG or IgA, was the best correlate of protection. The results of these studies suggest that both fCVEV1219 and gCVEV1219 should be evaluated further and considered for advancement as potential second-generation inactivated vaccine candidates for EEEV.     

Newcastle Disease Virus-Vectored Vaccines Expressing the Hemagglutinin or Neuraminidase Protein of H5N1 Highly Pathogenic Avian Influenza Virus Protect against Virus Challenge in Monkeys

H5N1 highly pathogenic avian influenza virus (HPAIV) causes periodic outbreaks in humans, resulting in severe infections with a high (60%) incidence of mortality. The circulating strains have low human-to-human transmissibility; however, widespread concerns exist that enhanced transmission due to mutations could lead to a global pandemic. We previously engineered Newcastle disease virus (NDV), an avian paramyxovirus, as a vector to express the HPAIV hemagglutinin (HA) protein, and we showed that this vaccine (NDV/HA) induced a high level of HPAIV-specific mucosal and serum antibodies in primates when administered through the respiratory tract. Here we developed additional NDV-vectored vaccines expressing either HPAIV HA in which the polybasic cleavage site was replaced with that from a low-pathogenicity strain of influenza virus [HA(RV)], in order to address concerns of enhanced vector replication or genetic exchange, or HPAIV neuraminidase (NA). The three vaccine viruses [NDV/HA, NDV/HA(RV), and NDV/NA] were administered separately to groups of African green monkeys by the intranasal/intratracheal route. An additional group of animals received NDV/HA by aerosol administration. Each of the vaccine constructs was highly restricted for replication, with only low levels of virus shedding detected in respiratory secretions. All groups developed high levels of neutralizing antibodies against homologous and heterologous strains of HPAIV and were protected against challenge with 2 × 10⁷ PFU of homologous HPAIV. Thus, needle-free, highly attenuated NDV-vectored vaccines expressing either HPAIV HA, HA(RV), or NA have been developed and demonstrated to be individually immunogenic and protective in a primate model of HPAIV infection. The finding that HA(RV) was protective indicates that it would be preferred for inclusion in a vaccine. The study also identified NA as an independent protective HPAIV antigen in primates. Furthermore, we demonstrated the feasibility of aerosol delivery of NDV-vectored vaccines.H5N1 highly pathogenic avian influenza virus (HPAIV) was first detected in human infections in 1997; previously, it had been found only in birds (11, 50). To date, this virus has been identified in 436 confirmed cases of human infection in 15 countries, 262 (60%) of which were fatal (75). The currently circulating H5N1 strains are characterized by low human-to-human transmissibility. This has been attributed, in part, to a preference for binding to α-2,3-linked sialic acids that are present in high concentrations throughout the avian respiratory tract but were thought to be found primarily in the lower human respiratory tract (57), although this explanation has been questioned (48, 49). It has also been observed that mutations in the PB2 subunit of the viral polymerase are necessary to confer the ability for the virus to be spread by aerosolized nasal droplets in ferrets (72). Whatever factors may be involved, there is widespread concern that the avian virus could mutate to enhance its transmissibility among humans, possibly resulting in a global pandemic (28, 50). For the avian H9N2 virus, which also has pandemic potential, it has been demonstrated that only five amino acid changes were sufficient for the virus to gain the ability to be spread by aerosolized nasal droplets in a ferret model (60). Thus, there is an urgent need for vaccines against HPAIV.Several vaccine strategies for HPAIV have been evaluated (reviewed in references 32 and 41), including inactivated and live attenuated vaccines. These efforts have been hampered by several factors. HPAIV strains are highly virulent for embryonated chicken eggs, the most widely used substrate for vaccine manufacture, and their rapid death following inoculation renders eggs unsuitable for efficient virus propagation. In addition, the major protective antigen, hemagglutinin (HA), administered either as a purified protein or in inactivated HPAIV virions, appears to be poorly immunogenic (69, 70). An additional factor complicating the development of HPAIV vaccines based on inactivated virus is the high cost and biohazard associated with HPAIV propagation, which must be done under enhanced biosafety level 3 (BSL-3) containment, although this problem might be addressed by the use of live attenuated reassortant influenza virus vaccines that contain the HPAIV glycoproteins on the background of an avirulent human influenza virus strain (24, 37). In addition, such reassortant strains might serve directly as live attenuated vaccines. Unfortunately, the latter approach may be limited by subtle and unpredictable incompatibility between the avian-origin glycoproteins and human-origin vaccine backgrounds acceptable for human use, which can result in overattenuation in vivo (24). There are also lingering concerns about the significant potential, with a live HPAIV vaccine, for reassortment between gene segments of the vaccine virus and circulating influenza virus strains, which might result in novel strains with unpredictable biological properties (63).We and others have been evaluating Newcastle disease virus (NDV) as a general human vaccine vector for emerging pathogens, including H5N1 HPAIV (7, 18-20, 29). NDV is an avian paramyxovirus that is antigenically unrelated to common human pathogens; hence, its use in humans should not be affected by host immunity to common pathogens. The many naturally occurring strains of NDV can be categorized into three pathotypes based on virulence in chickens: velogenic strains, causing severe disease with high mortality; mesogenic strains, causing disease of intermediate severity with low mortality; and lentogenic strains, causing mild or inapparent infections (reviewed in reference 2). Lentogenic, and sometimes mesogenic, strains of NDV are in wide use as live attenuated vaccines against velogenic NDV in poultry (2). When mesogenic or lentogenic NDV was administered to the respiratory tracts of nonhuman primates as a model for the immunization of humans, the virus was highly attenuated for replication, was shed only at low titers, appeared to remain restricted to the respiratory tract, and was highly immunogenic for the expressed foreign antigen (7). We recently demonstrated that a mesogenic strain of NDV expressing the HA protein of H5N1 HPAIV (NDV/HA) elicited high titers of neutralizing antibodies in serum following combined intranasal (i.n.) and intratracheal (i.t.) delivery in a nonhuman primate model (20). Vaccination of mice with a similar NDV-vectored vaccine protected them from HPAIV challenge (29). However, results obtained with mice do not reliably predict the efficacy of an influenza virus vaccine for human use, due to the pathophysiological and phylogenetic differences between mice and humans (71). In particular, mice may produce a potent immune response to HPAIV vaccines (64) that may not be reproduced in clinical trials (38). These considerations are especially important for a vaccine based on a live viral vector platform, since its immunogenicity, and therefore its protective efficacy, is directly linked to replication, which can differ greatly in various experimental animals versus humans (reviewed in references 6 and 9). Therefore, the protective efficacy of NDV-based vaccines against HPAIV challenge in nonhuman primate models—the closest model to humans—has remained unknown.The protease recognition sequence of the HA protein is one of the major determinants of avian influenza virus pathogenicity (62). HPAIV strains have a “polybasic” cleavage site, containing multiple basic amino acids, that is readily cleaved by ubiquitous intracellular subtilisin-like proteases, facilitating the replication and spread of the virus. In contrast, the HA cleavage site of low-pathogenicity strains contains fewer basic amino acids and depends on secretory trypsin-like proteases found in the respiratory and enteric tracts, resulting in more-localized infections (30, 62). The presence of a polybasic cleavage site in the H5 HA of any live vaccine raises some concern about the possibility of genetic exchange with circulating strains of influenza virus. It should be noted that genetic exchange involving paramyxoviruses is a rare event (14) that has been documented only once (61). However, elimination of the polybasic HA cleavage site would mitigate the effects of even this rare possibility of genetic exchange. Another concern was based on our previous finding that the HPAIV H5 HA protein is incorporated into the NDV envelope as a trimer (20), consistent with its presence in a functional form. While we previously showed that this did not enhance the pathogenicity of the NDV/HA recombinant in chickens (20), we could not rule out the possibility that it might confer an altered tropism on the NDV/HA virus in other systems. For example, a recombinant parainfluenza virus type 3 expressing the Ebola virus glycoprotein incorporated the foreign protein into its envelope, allowing cellular attachment and fusion of the vaccine virus independently of the vector''s own envelope glycoproteins (10).In addition to the HA protein, the neuraminidase (NA) protein is also present on the surfaces of influenza virus-infected cells and virions. Antibodies specific for NA are not thought to interfere with the initial viral attachment and penetration of host cells (36, 40, 54). However, NA-specific antibodies prevent the release of virus from infected cells, thereby decreasing viral spread (35), and they increase resistance to viral infection in humans (40, 47, 54). They also provide at least some protection against viruses bearing homologous or heterologous NA proteins of the same subtype in a mouse model (12, 56). NA also appears to evolve at a lower rate than HA, suggesting that NA-specific antibodies may provide broader protection than a vaccine utilizing HA alone (39). Therefore, it was important to assess the immunogenicity and protective efficacy of the HPAIV NA independently of those of HA, which has not previously been done in a human or nonhuman primate model.     

鸡OV启动子表达HA对禽流感病毒攻击提供完全保护

鸡卵清蛋白(ovalbumin,OV)基因5'调控序列是构建鸡输卵管生物反应器的首选调控元件。以EGFP为报告基因,构建OV启动子真核表达载体,转染原代输卵管上皮细胞和CHO细胞,筛选得到1.1kb的高效OV启动子。构建1.1kb OV启动子表达H5N1亚型禽流感病毒HA蛋白真核表达载体pOV_(1.1k)-HA,转染CHO细胞。PCR、RT-PCR鉴定结果证明HA基因整合至CHO细胞基因组,并进行转录;SDS-PAGE、Western blot及HA试验结果证明HA蛋白在CHO细胞内的表达,并具有免疫反应性和血凝活性。以纯化的HA蛋白免疫4周龄SPF鸡,2周后加强免疫一次,加强免疫3周HI抗体水平为6.3log2;以10~6EID_(50)H5N1(A/Goose/Guangdong/1/96)亚型禽流感病毒鼻腔接种SPF鸡,免疫组100%存活,无排毒现象,对照组100%死亡。结果表明,筛选的1.1kb OV启动子可有效驱动HA蛋白表达,表达的HA蛋白免疫SPF鸡对禽流感病毒攻击提供完全保护;为鸡输卵管生物反应器表达保护性抗原和珍贵药物蛋白奠定了基础。     

New Orf Virus (Parapoxvirus) Recombinant Expressing H5 Hemagglutinin Protects Mice against H5N1 and H1N1 Influenza A Virus

Previously we demonstrated the versatile utility of the Parapoxvirus Orf virus (ORFV) as a vector platform for the development of potent recombinant vaccines. In this study we present the generation of new ORFV recombinants expressing the hemagglutinin (HA) or nucleoprotein (NP) of the highly pathogenic avian influenza virus (HPAIV) H5N1. Correct foreign gene expression was examined in vitro by immunofluorescence, Western blotting and flow cytometry. The protective potential of both recombinants was evaluated in the mouse challenge model. Despite adequate expression of NP, the recombinant D1701-V-NPh5 completely failed to protect mice from lethal challenge. However, the H5 HA-expressing recombinant D1701-V-HAh5n mediated solid protection in a dose-dependent manner. Two intramuscular (i.m.) injections of the HA-expressing recombinant protected all animals from lethal HPAIV infection without loss of body weight. Notably, the immunized mice resisted cross-clade H5N1 and heterologous H1N1 (strain PR8) influenza virus challenge. In vivo antibody-mediated depletion of CD4-positive and/or CD8-posititve T-cell subpopulations during immunization and/or challenge infection implicated the relevance of CD4-positive T-cells for induction of protective immunity by D1701-V-HAh5n, whereas the absence of CD8-positive T-cells did not significantly influence protection. In summary, this study validates the potential of the ORFV vectored vaccines also to combat HPAIV.