Evaluation of MDCK Cell-Derived Influenza H7N9 Vaccine Candidates in Ferrets

Avian-origin influenza A (H7N9) viruses emerged as human pathogens in China in early 2013 and have killed >100 persons. Influenza vaccines are mainly manufactured using egg-based technology which could not meet the surging demand during influenza pandemics. In this study, we evaluated cell-based influenza H7N9 vaccines in ferrets. An egg-derived influenza H7N9 reassortant vaccine virus was adapted in MDCK cells. Influenza H7N9 whole virus vaccine antigen was manufactured using a microcarrier-based culture system. Immunogenicity and protection of the vaccine candidates with three different formulations (300μg aluminum hydroxide, 1.5μg HA, and 1.5μg HA plus 300μg aluminum hydroxide) were evaluated in ferrets. In ferrets receiving two doses of vaccination, geometric mean titers of hemagglutination (HA) inhibition and neutralizing antibodies were <10 and <40 for the control group (adjuvant only), 17 and 80 for the unadjuvanted (HA only) group, and 190 and 640 for the adjuvanted group (HA plus adjuvant), respectively. After challenge with wild-type influenza H7N9 viruses, virus titers in respiratory tracts of the adjuvanted group were significantly lower than that in the control, and unadjuvanted groups. MDCK cell-derived influenza H7N9 whole virus vaccine candidate is immunogenic and protective in ferrets and clinical development is highly warranted.     

A new generation of modified live-attenuated avian influenza viruses using a two-strategy combination as potential vaccine candidates

In light of the recurrent outbreaks of low pathogenic avian influenza (LPAI) and highly pathogenic avian influenza (HPAI), there is a pressing need for the development of vaccines that allow rapid mass vaccination. In this study, we introduced by reverse genetics temperature-sensitive mutations in the PB1 and PB2 genes of an avian influenza virus, A/Guinea Fowl/Hong Kong/WF10/99 (H9N2) (WF10). Further genetic modifications were introduced into the PB1 gene to enhance the attenuated (att) phenotype of the virus in vivo. Using the att WF10 as a backbone, we substituted neuraminidase (NA) for hemagglutinin (HA) for vaccine purposes. In chickens, a vaccination scheme consisting of a single dose of an att H7N2 vaccine virus at 2 weeks of age and subsequent challenge with the wild-type H7N2 LPAI virus resulted in complete protection. We further extended our vaccination strategy against the HPAI H5N1. In this case, we reconstituted an att H5N1 vaccine virus, whose HA and NA genes were derived from an Asian H5N1 virus. A single-dose immunization in ovo with the att H5N1 vaccine virus in 18-day-old chicken embryos resulted in more than 60% protection for 4-week-old chickens and 100% protection for 9- to 12-week-old chickens. Boosting at 2 weeks posthatching provided 100% protection against challenge with the HPAI H5N1 virus for chickens as young as 4 weeks old, with undetectable virus shedding postchallenge. Our results highlight the potential of live att avian influenza vaccines for mass vaccination in poultry.     

Contributions of the Avian Influenza Virus HA,NA, and M2 Surface Proteins to the Induction of Neutralizing Antibodies and Protective Immunity

Highly pathogenic avian influenza virus (HPAIV) subtype H5N1 causes severe disease and mortality in poultry. Increased transmission of H5N1 HPAIV from birds to humans is a serious threat to public health. We evaluated the individual contributions of each of the three HPAIV surface proteins, namely, the hemagglutinin (HA), the neuraminidase (NA), and the M2 proteins, to the induction of HPAIV-neutralizing serum antibodies and protective immunity in chickens. Using reverse genetics, three recombinant Newcastle disease viruses (rNDVs) were engineered, each expressing the HA, NA, or M2 protein of H5N1 HPAIV. Chickens were immunized with NDVs expressing a single antigen (HA, NA, and M2), two antigens (HA+NA, HA+M2, and NA+M2), or three antigens (HA+NA+M2). Immunization with HA or NA induced high titers of HPAIV-neutralizing serum antibodies, with the response to HA being greater, thus identifying HA and NA as independent neutralization antigens. M2 did not induce a detectable neutralizing serum antibody response, and inclusion of M2 with HA or NA reduced the magnitude of the response. Immunization with HA alone or in combination with NA induced complete protection against HPAIV challenge. Immunization with NA alone or in combination with M2 did not prevent death following challenge, but extended the time period before death. Immunization with M2 alone had no effect on morbidity or mortality. Thus, there was no indication that M2 is immunogenic or protective. Furthermore, inclusion of NA in addition to HA in a vaccine preparation for chickens may not enhance the high level of protection provided by HA.Avian influenza (AI) is an economically important disease of poultry worldwide. Avian influenza virus (AIV) belongs to the genus Influenzavirus A under the family Orthomyxoviridae. The genome of AIV consists of eight segments of single-stranded, negative-sense RNA that codes for 11 proteins (PB2, PB1, PB1-F2, PA, HA, NP, NA, M1, M2, NS1, and NS2/NEP). The genome is surrounded by the viral envelope that has two glycoprotein spikes on its outer surface, hemagglutinin (HA) and neuraminidase (NA). The HA spikes have receptor binding and fusion functions, and NA spikes have receptor-destroying activity. The envelope also contains a third integral membrane protein, M2, which is exposed on the outer surface and functions as an ion channel, essential for uncoating. The AIV surface glycoproteins are antigenically variable and are serologically divided into 16 HA (H1 to H16) and 9 NA (N1 to N9) subtypes, whereas the nonglycosylated surface protein M2 is highly conserved (9, 43). On the basis of severity of disease in poultry, AIV strains are also classified into low-pathogenic (LP) and highly pathogenic (HP) categories. Historically, highly pathogenic avian influenza viruses (HPAIV) of subtypes H5 and H7 have caused severe disease and mortality in poultry. Recent HPAIV subtype H5N1 infections have resulted in the culling or death of more than 500 million poultry in more than 62 countries (27). Since 1997, HPAIV strains of subtype H5N1 have been found to cause disease in humans. To date, this virus has caused 436 confirmed human infections. Of these infections, 262 (60%) were fatal. Hence, HPAIV has become a major threat to both animals and humans (45). The World Organisation of Animal Health (OIE) recommends the control of HPAIV at its poultry source to decrease the viral load in susceptible avian species, thereby decreasing the risk of transmission to humans (31). The traditional method of control of HPAI has been stamping out infected flocks, which is still used in many countries, including the United States. But, due to economic reasons, culling of infected flocks is no longer considered a practical method for the control of AI in either developed or developing countries. Vaccination has been recommended by the OIE to control AI (31). Several vaccination strategies, including inactivated and live attenuated vaccines, have been evaluated for HPAIV (28). Inactivated vaccines are not commonly used because of the high cost and the difficulty in “differentiating infected from vaccinated animals” (DIVA). Live attenuated vaccines are not used because of the concern that the vaccine viruses may, through either mutation or genetic reassortment with circulating strains, become virulent (1). To overcome these difficulties, recombinant DNA technology was used to generate vectored, subunit, or DNA vaccines. Although several of these vaccines have been shown experimentally to protect against AIV, Newcastle disease virus (NDV)-vectored vaccines have shown the most promising results and also have the advantage of being bivalent vaccines against both NDV and AIV (11, 25, 32, 42). Furthermore, NDV-vectored vaccines have also been evaluated in primates with promising results (6). Newcastle disease (ND) is an economically important disease in poultry worldwide. The causative agent (NDV) is a nonsegmented, negative-strand RNA virus belonging to the genus Avulavirus in the family Paramyxoviridae. NDV strains vary greatly in virulence. Virulent NDV strains cause a severe respiratory and neurologic disease in poultry worldwide. Naturally occurring avirulent NDV strains are routinely used to control ND in many parts of world (30).We recently evaluated recombinant NDV (rNDV) expressing the HA protein of an H5N1 HPAIV vaccine (rNDV-HA) in chickens (25). Chickens immunized with rNDV-HA produced NDV- and HPAIV H5-specific antibodies and were protected against clinical disease after challenge with virulent NDV or HPAIV. Furthermore, shedding of the challenge virus was not observed, indicating complete protection. Our results demonstrated that rNDV-HA is a suitable bivalent vaccine against NDV and AIV (25). To date, all NDV-vectored vaccine studies in chickens have used HA genes derived from various HPAIV strains (11, 25, 32, 42). However, in addition to the HA protein, the envelope of AIV contains two other proteins (NA and M2) on its outer surface. Although antibodies to NA are thought not to play any role in viral attachment and penetration of the host cell, they prevent the release of virus from infected cells (20) and increase overall resistance to AIV infection in humans (37). The NA gene is thought to evolve at a lower rate than the HA gene, indicating that NA-specific antibodies may increase the breadth of protection of the HA-specific antibodies (19). The other surface protein, M2, functions as an ion channel protein and also as a target for anti-HPAIV drugs. The role of M2 protein in the induction of HPAIV-neutralizing antibodies and protective immunity is not well understood. Antibodies induced by the M2e peptide corresponding to the N-terminal 24-amino-acid ectodomain (the portion present on the virus surface) displayed broad protection against influenza A viruses of both homologous (H1N1) and heterologous (H3N1) strains in vitro and in vivo (7). However, the role of entire length of the M2 protein of AIV in induction of neutralizing antibodies and protective immunity against highly pathogenic H5N1 influenza virus in chickens has not been directly evaluated. The M2 protein is conserved among all influenza A viruses and is therefore considered an attractive target for a “universal” vaccine (8). Antibodies to HA protein alone can protect against lethal AIV challenges; the inclusion of other surface proteins in the vaccine regimen may improve the protective efficacy.In the present study, we examined the relative contribution of each of the three HPAIV surface proteins (HA, NA, and M2) to induction of neutralizing antibodies and protective immunity in chickens. Recombinant NDV vectors were constructed that individually expressed each of the three HPAIV surface proteins. They were used to immunize chickens either individually or in different possible combinations. Evaluation of the relative neutralization titers of serum antibody, shedding of challenge virus, and protection against lethal HPAIV challenge conferred by each of the NDV-vectored HPAIV surface proteins showed that HA glycoprotein was the major contributor to induction of neutralizing antibodies and protective immunity, followed by NA protein, which conferred partial protection. The M2 protein neither induced a detectable level of serum-neutralizing antibodies nor protected chickens from the HPAIV lethal challenge.     

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.     

Recombinant Parainfluenza Virus 5 Vaccine Encoding the Influenza Virus Hemagglutinin Protects against H5N1 Highly Pathogenic Avian Influenza Virus Infection following Intranasal or Intramuscular Vaccination of BALB/c Mice

New approaches for vaccination to prevent influenza virus infection are needed. Emerging viruses, such as the H5N1 highly pathogenic avian influenza (HPAI) virus, pose not only pandemic threats but also challenges in vaccine development and production. Parainfluenza virus 5 (PIV5) is an appealing vector for vaccine development, and we have previously shown that intranasal immunization with PIV5 expressing the hemagglutinin from influenza virus was protective against influenza virus challenge (S. M. Tompkins, Y. Lin, G. P. Leser, K. A. Kramer, D. L. Haas, E. W. Howerth, J. Xu, M. J. Kennett, J. E. Durbin, R. A. Tripp, R. A. Lamb, and B. He, Virology 362:139–150, 2007). While intranasal immunization is an appealing approach, PIV5 may have the potential to be utilized in other formats, prompting us to test the efficacy of rPIV5-H5, which encodes the HA from H5N1 HPAI virus, in different vaccination schemes. In the BALB/c mouse model, a single intramuscular or intranasal immunization with a live rPIV5-H5 (ZL46) rapidly induced robust neutralizing serum antibody responses and protected against HPAI challenge, although mucosal IgA responses primed by intranasal immunization more effectively controlled virus replication in the lung. The rPIV5-H5 vaccine incorporated the H5 HA into the virion, so we tested the efficacy of an inactivated form of the vaccine. Inactivated rPIV5-H5 primed neutralizing serum antibody responses and controlled H5N1 virus replication; however, similar to other H5 antigen vaccines, it required a booster immunization to prime protective immune responses. Taken together, these results suggest that rPIV5-HA vaccines and H5-specific vaccines in particular can be utilized in multiple formats and by multiple routes of administration. This could avoid potential contraindications based on intranasal administration alone and provide opportunities for broader applications with the use of a single vaccine vector.     

Induction of heterosubtypic immunity to influenza virus by intranasal immunization

Recovery from live influenza virus infection is known to induce heterosubtypic immunity. In contrast, immunity induced by inactivated vaccines is predominantly subtype specific. In this study, we investigated the heterosubtypic protective immunity induced by inactivated influenza virus. Intranasal immunization of mice with inactivated influenza virus A/PR8 (H1N1) provided complete protection against the homologous virus and a drift virus within the same subtype, A/WSN (H1N1), but not against the heterosubtypic virus A/Philippines (H3N2). However, coadministration of inactivated virus with cholera toxin as an adjuvant conferred complete heterosubtypic protection, without observed illness, even under conditions of CD4⁺ or CD8⁺ T-cell depletion. Analysis of immune correlates prior to challenge and postchallenge indicated that humoral immune responses with cross-neutralizing activity in lungs and in sera play a major role in conferring protective immunity against heterosubtypic challenge. This study has significant implications for developing broadly cross-reactive vaccines against newly emerging pathogenic influenza viruses.     

Vaccination against Human Influenza A/H3N2 Virus Prevents the Induction of Heterosubtypic Immunity against Lethal Infection with Avian Influenza A/H5N1 Virus

Annual vaccination against seasonal influenza viruses is recommended for certain individuals that have a high risk for complications resulting from infection with these viruses. Recently it was recommended in a number of countries including the USA to vaccinate all healthy children between 6 and 59 months of age as well. However, vaccination of immunologically naïve subjects against seasonal influenza may prevent the induction of heterosubtypic immunity against potentially pandemic strains of an alternative subtype, otherwise induced by infection with the seasonal strains.Here we show in a mouse model that the induction of protective heterosubtypic immunity by infection with a human A/H3N2 influenza virus is prevented by effective vaccination against the A/H3N2 strain. Consequently, vaccinated mice were no longer protected against a lethal infection with an avian A/H5N1 influenza virus. As a result H3N2-vaccinated mice continued to loose body weight after A/H5N1 infection, had 100-fold higher lung virus titers on day 7 post infection and more severe histopathological changes than mice that were not protected by vaccination against A/H3N2 influenza.The lack of protection correlated with reduced virus-specific CD8+ T cell responses after A/H5N1 virus challenge infection. These findings may have implications for the general recommendation to vaccinate all healthy children against seasonal influenza in the light of the current pandemic threat caused by highly pathogenic avian A/H5N1 influenza viruses.     

Evaluation of the efficacy of a pre-pandemic H5N1 vaccine (MG1109) in mouse and ferret models

The threat of a highly pathogenic avian influenza (HPAI) H5N1 virus causing the next pandemic remains a major concern. In this study, we evaluated the immunogenicity and efficacy of an inactivated whole-virus H5N1 pre-pandemic vaccine (MG1109) formulated by Green Cross Co., Ltd containing the hemagglutinin (HA) and neuraminidase (NA) genes of the clade 1 A/Vietnam/1194/04 virus in the backbone of A/Puerto Rico/8/34 (RgVietNam/04xPR8/34). Administration of the MG1109 vaccine (2-doses) in mice and ferrets elicited high HI and SN titers in a dose-dependent manner against the homologous (RgVietNam/04xPR8/34) and various heterologous H5N1 strains, (RgKor/W149/06xPR8/34, RgCambodia/04xPR8/34, RgGuangxi/05xPR8/34), including a heterosubtypic H5N2 (A/Aquatic bird/orea/W81/05) virus. However, efficient cross-reactivity was not observed against heterosubtypic H9N2 (A/Ck/Korea/H0802/08) and H1N1 (PR/8/34) viruses. Mice immunized with 1.9 μg HA/dose of MG1109 were completely protected from lethal challenge with heterologous wild-type HPAI H5N1 A/EM/Korea/W149/06 (clade 2.2) and mouse-adapted H5N2 viruses. Furthermore, ferrets administered at least 3.8 μg HA/dose efficiently suppressed virus growth in the upper respiratory tract and lungs. Vaccinated mice and ferrets also demonstrated attenuation of clinical disease signs and limited virus spread to other organs. Thus, this vaccine provided immunogenic responses in mouse and ferret models even against challenge with heterologous HPAI H5N1 and H5N2 viruses. Since the specific strain of HPAI H5N1 virus that would potentially cause the next outbreak is unknown, pre-pandemic vaccine preparation that could provide cross-protection against various H5 strains could be a useful approach in the selection of promising candidate vaccines in the future.     

Intranasal H5N1 Vaccines,Adjuvanted with Chitosan Derivatives,Protect Ferrets against Highly Pathogenic Influenza Intranasal and Intratracheal Challenge

We investigated the protective efficacy of two intranasal chitosan (CSN and TM-CSN) adjuvanted H5N1 Influenza vaccines against highly pathogenic avian Influenza (HPAI) intratracheal and intranasal challenge in a ferret model.Six groups of 6 ferrets were intranasally vaccinated twice, 21 days apart, with either placebo, antigen alone, CSN adjuvanted antigen, or TM-CSN adjuvanted antigen. Homologous and intra-subtypic antibody cross-reacting responses were assessed. Ferrets were inoculated intratracheally (all treatments) or intranasally (CSN adjuvanted and placebo treatments only) with clade 1 HPAI A/Vietnam/1194/2004 (H5N1) virus 28 days after the second vaccination and subsequently monitored for morbidity and mortality outcomes. Clinical signs were assessed and nasal as well as throat swabs were taken daily for virology. Samples of lung tissue, nasal turbinates, brain, and olfactory bulb were analysed for the presence of virus and examined for histolopathological findings.In contrast to animals vaccinated with antigen alone, the CSN and TM-CSN adjuvanted vaccines induced high levels of antibodies, protected ferrets from death, reduced viral replication and abrogated disease after intratracheal challenge, and in the case of CSN after intranasal challenge. In particular, the TM-CSN adjuvanted vaccine was highly effective at eliciting protective immunity from intratracheal challenge; serologically, protective titres were demonstrable after one vaccination. The 2-dose schedule with TM-CSN vaccine also induced cross-reactive antibodies to clade 2.1 and 2.2 H5N1 viruses. Furthermore ferrets immunised with TM-CSN had no detectable virus in the respiratory tract or brain, whereas there were signs of virus in the throat and lungs, albeit at significantly reduced levels, in CSN vaccinated animals.This study demonstrated for the first time that CSN and in particular TM-CSN adjuvanted intranasal vaccines have the potential to protect against significant mortality and morbidity arising from infection with HPAI H5N1 virus.     

Heterosubtypic Neutralizing Monoclonal Antibodies Cross-Protective against H5N1 and H1N1 Recovered from Human IgM+ Memory B Cells

Background

The hemagglutinin (HA) glycoprotein is the principal target of protective humoral immune responses to influenza virus infections but such antibody responses only provide efficient protection against a narrow spectrum of HA antigenic variants within a given virus subtype. Avian influenza viruses such as H5N1 are currently panzootic and pose a pandemic threat. These viruses are antigenically diverse and protective strategies need to cross protect against diverse viral clades. Furthermore, there are 16 different HA subtypes and no certainty the next pandemic will be caused by an H5 subtype, thus it is important to develop prophylactic and therapeutic interventions that provide heterosubtypic protection.

Methods and Findings

Here we describe a panel of 13 monoclonal antibodies (mAbs) recovered from combinatorial display libraries that were constructed from human IgM⁺ memory B cells of recent (seasonal) influenza vaccinees. The mAbs have broad heterosubtypic neutralizing activity against antigenically diverse H1, H2, H5, H6, H8 and H9 influenza subtypes. Restriction to variable heavy chain gene IGHV1-69 in the high affinity mAb panel was associated with binding to a conserved hydrophobic pocket in the stem domain of HA. The most potent antibody (CR6261) was protective in mice when given before and after lethal H5N1 or H1N1 challenge.

Conclusions

The human monoclonal CR6261 described in this study could be developed for use as a broad spectrum agent for prophylaxis or treatment of human or avian influenza infections without prior strain characterization. Moreover, the CR6261 epitope could be applied in targeted vaccine strategies or in the design of novel antivirals. Finally our approach of screening the IgM⁺ memory repertoire could be applied to identify conserved and functionally relevant targets on other rapidly evolving pathogens.     

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.     

A Live Attenuated H7N7 Candidate Vaccine Virus Induces Neutralizing Antibody That Confers Protection from Challenge in Mice,Ferrets, and Monkeys

A live attenuated H7N7 candidate vaccine virus was generated by reverse genetics using the modified hemagglutinin (HA) and neuraminidase (NA) genes of highly pathogenic (HP) A/Netherlands/219/03 (NL/03) (H7N7) wild-type (wt) virus and the six internal protein genes of the cold-adapted (ca) A/Ann Arbor/6/60 ca (AA ca) (H2N2) virus. The reassortant H7N7 NL/03 ca vaccine virus was temperature sensitive and attenuated in mice, ferrets, and African green monkeys (AGMs). Intranasal (i.n.) administration of a single dose of the H7N7 NL/03 ca vaccine virus fully protected mice from lethal challenge with homologous and heterologous H7 viruses from Eurasian and North American lineages. Two doses of the H7N7 NL/03 ca vaccine induced neutralizing antibodies in serum and provided complete protection from pulmonary replication of homologous and heterologous wild-type H7 challenge viruses in mice and ferrets. One dose of the H7N7 NL/03 ca vaccine elicited an antibody response in one of three AGMs that was completely protected from pulmonary replication of the homologous wild-type H7 challenge virus. The contribution of CD8⁺ and/or CD4⁺ T cells to the vaccine-induced protection of mice was evaluated by T-cell depletion; T lymphocytes were not essential for the vaccine-induced protection from lethal challenge with H7 wt viruses. Additionally, passively transferred neutralizing antibody induced by the H7N7 NL/03 ca virus protected mice from lethality following challenge with H7 wt viruses. The safety, immunogenicity, and efficacy of the H7N7 NL/03 ca vaccine virus in mice, ferrets, and AGMs support the evaluation of this vaccine virus in phase I clinical trials.Highly pathogenic avian influenza (HPAI) is a disease of poultry that is caused by H5 or H7 avian influenza viruses and is associated with up to 100% mortality (2). Influenza A H7 subtype viruses from both Eurasian and North American lineages have resulted in more than 100 cases of human infection since 2002 in the Netherlands, Italy, Canada, the United Kingdom, and the United States. These cases include outbreaks of HPAI H7N7 virus in the Netherlands in 2003 that resulted in more than 80 cases of human infection and one fatality; HPAI H7N3 virus in British Columbia, Canada, in 2004 that resulted in two cases of conjunctivitis; a cluster of human infections of low-pathogenicity avian influenza (LPAI) H7N2 virus in the United Kingdom in 2007 that resulted in several cases of influenza-like illness and conjunctivitis; and a single case of respiratory infection in New York in 2003 (3-6, 17, 27).Due to an unprecedented geographic spread of H5 subtype viruses since 2003 and the continued occurrence of sporadic cases of H5N1 infections in humans, much emphasis has been placed on the pandemic threat posed by H5 subtype viruses. However, H7 subtype viruses also have significant pandemic potential. Humans are immunologically naïve to the H7 avian influenza viruses (16), and LPAI H7 subtype viruses circulating in domestic poultry and wild birds in Eurasia and North America have the potential to evolve and acquire an HP phenotype either by accumulating mutations or by recombination at the hemagglutinin (HA) cleavage site resulting in a highly cleavable HA that is a virulence motif in poultry (30, 33, 34). Recent work also suggests that contemporary North American lineage H7 subtype viruses, isolated in 2002 to 2003, are partially adapted to recognize α2-6-linked sialic acids, which are the receptors preferred by human influenza viruses and are preferentially found in the human upper respiratory tract (7). Moreover, coinfection and genetic reassortment of RNA genomes between H7 avian influenza viruses and human influenza viruses, including the seasonal H1N1 and H3N2 and pandemic H1N1 viruses, could result in the generation of reassortant viruses with the capacity to efficiently transmit among people and result in a pandemic. Domesticated birds may serve as important intermediate hosts for the transmission of wild-bird influenza viruses to humans, as may pigs, as evidenced by human infections with swine-origin 2009 pandemic H1N1 influenza virus throughout the world.Vaccination is the most effective method for the prevention of influenza. However, technical limitations result in delays in the rapid generation and availability of a strain-specific vaccine against an emerging pandemic virus. The emergence of antigenically distinct virus clades poses a substantial challenge for the design of vaccines against H5N1 viruses because of the possible need for clade-specific vaccines (1). Similar challenges are present for the generation of H7 subtype vaccine candidates, because antigenically distinct H7 subtype viruses, including North American lineage H7N2 and H7N3 and Eurasian lineage H7N7 and H7N3 viruses, have caused human disease. The successful control of H7 influenza virus in poultry has been achieved by stamping out and by vaccination of poultry (9). Vaccines for human use against both lineages of H7 influenza virus are under development, and candidate vaccines have been evaluated in preclinical and clinical studies (14, 23, 29, 42).We have previously analyzed the antigenic relatedness among H7 viruses from Eurasian and North American lineages using postinfection mouse and ferret sera (22). Among 10 H7 viruses tested, A/Netherlands/219/03 (H7N7) virus induced the most broadly cross-neutralizing antibodies (Abs) (22). Based on the phylogenetic relationships and its ability to induce broadly cross-neutralizing antibodies in mice and ferrets, we selected the A/Netherlands/219/03 (NL/03) (H7N7) virus from the Eurasian lineage for vaccine development. We used reverse genetics to generate a live attenuated cold-adapted (ca) H7N7 candidate vaccine virus bearing a modified HA, a wild-type (wt) neuraminidase (NA) gene from the NL/03 wt virus, and the six internal protein gene segments from the cold-adapted (ca) influenza A virus vaccine donor strain, A/Ann Arbor/6/60 ca (AA ca) (H2N2). The immunogenicity and protective efficacy against challenge with HP and LP H7 viruses from the Eurasian and North American lineages of the reassortant H7N7 NL03/AA ca vaccine virus were evaluated in mice, ferrets, and African green monkeys (AGMs).     

A Trivalent Virus-Like Particle Vaccine Elicits Protective Immune Responses against Seasonal Influenza Strains in Mice and Ferrets

There is need for improved human influenza vaccines, particularly for older adults who are at greatest risk for severe disease, as well as to address the continuous antigenic drift within circulating human subtypes of influenza virus. We have engineered an influenza virus-like particle (VLP) as a new generation vaccine candidate purified from the supernatants of Sf9 insect cells following infection by recombinant baculoviruses to express three influenza virus proteins, hemagglutinin (HA), neuraminidase (NA), and matrix 1 (M1). In this study, a seasonal trivalent VLP vaccine (TVV) formulation, composed of influenza A H1N1 and H3N2 and influenza B VLPs, was evaluated in mice and ferrets for the ability to elicit antigen-specific immune responses. Animals vaccinated with the TVV formulation had hemagglutination-inhibition (HAI) antibody titers against all three homologous influenza virus strains, as well as HAI antibodies against a panel of heterologous influenza viruses. HAI titers elicited by the TVV were statistically similar to HAI titers elicited in animals vaccinated with the corresponding monovalent VLP. Mice vaccinated with the TVV had higher level of influenza specific CD8+ T cell responses than a commercial trivalent inactivated vaccine (TIV). Ferrets vaccinated with the highest dose of the VLP vaccine and then challenged with the homologous H3N2 virus had the lowest titers of replicating virus in nasal washes and showed no signs of disease. Overall, a trivalent VLP vaccine elicits a broad array of immunity and can protect against influenza virus challenge.     

Human Cytotoxic T-Lymphocyte Repertoire to Influenza A Viruses

The murine CD8⁺ cytotoxic-T-lymphocyte (CTL) repertoire appears to be quite limited in response to influenza A viruses. The CTL responses to influenza A virus in humans were examined to determine if the CTL repertoire is also very limited. Bulk cultures revealed that a number of virus proteins were recognized in CTL assays. CTL lines were isolated from three donors for detailed study and found to be specific for epitopes on numerous influenza A viral proteins. Eight distinct CD8⁺ CTL lines were isolated from donor 1. The proteins recognized by these cell lines included the nucleoprotein (NP), matrix protein (M1), nonstructural protein 1 (NS1), polymerases (PB1 and PB2), and hemagglutinin (HA). Two CD4⁺ cell lines, one specific for neuraminidase (NA) and the other specific for M1, were also characterized. These CTL results were confirmed by precursor frequency analysis of peptide-specific gamma interferon-producing cells detected by ELISPOT. The epitopes recognized by 6 of these 10 cell lines have not been previously described; 8 of the 10 cell lines were cross-reactive to subtype H1N1, H2N2, and H3N2 viruses, 1 cell line was cross-reactive to subtypes H1N1 and H2N2, and 1 cell line was subtype H1N1 specific. A broad CTL repertoire was detected in the two other donors, and cell lines specific for the NP, NA, HA, M1, NS1, and M2 viral proteins were isolated. These findings indicate that the human memory CTL response to influenza A virus is broadly directed to epitopes on a wide variety of proteins, unlike the limited response observed following infection of mice.     

Induction of heterosubtypic cross-protection against influenza by a whole inactivated virus vaccine: the role of viral membrane fusion activity

Background

The inability of seasonal influenza vaccines to effectively protect against infection with antigenically drifted viruses or newly emerging pandemic viruses underlines the need for development of cross-reactive influenza vaccines that induce immunity against a variety of virus subtypes. Therefore, potential cross-protective vaccines, e.g., whole inactivated virus (WIV) vaccine, that can target conserved internal antigens such as the nucleoprotein (NP) and/or matrix protein (M1) need to be explored.

Methodology/Principal Findings

In the current study we show that a WIV vaccine, through induction of cross-protective cytotoxic T lymphocytes (CTLs), protects mice from heterosubtypic infection. This protection was abrogated after depletion of CD8+ cells in vaccinated mice, indicating that CTLs were the primary mediators of protection. Previously, we have shown that different procedures used for virus inactivation influence optimal activation of CTLs by WIV, most likely by affecting the membrane fusion properties of the virus. Specifically, inactivation with formalin (FA) severely compromises fusion activity of the virus, while inactivation with β-propiolactone (BPL) preserves fusion activity. Here, we demonstrate that vaccination of mice with BPL-inactivated H5N1 WIV vaccine induces solid protection from lethal heterosubtypic H1N1 challenge. By contrast, vaccination with FA-inactivated WIV, while preventing death after lethal challenge, failed to protect against development of disease and severe body weight loss. Vaccination with BPL-inactivated WIV, compared to FA-inactivated WIV, induced higher levels of specific CD8+ T cells in blood, spleen and lungs, and a higher production of granzyme B in the lungs upon H1N1 virus challenge.

Conclusion/Significance

The results underline the potential use of WIV as a cross-protective influenza vaccine candidate. However, careful choice of the virus inactivation procedure is important to retain membrane fusion activity and full immunogenicity of the vaccine.     

Cross-protection against lethal H5N1 challenge in ferrets with an adjuvanted pandemic influenza vaccine

Background

Unprecedented spread between birds and mammals of highly pathogenic avian influenza viruses (HPAI) of the H5N1 subtype has resulted in hundreds of human infections with a high fatality rate. This has highlighted the urgent need for the development of H5N1 vaccines that can be produced rapidly and in sufficient quantities. Potential pandemic inactivated vaccines will ideally induce substantial intra-subtypic cross-protection in humans to warrant the option of use, either prior to or just after the start of a pandemic outbreak. In the present study, we evaluated a split H5N1 A/H5N1/Vietnam/1194/04, clade 1 candidate vaccine, adjuvanted with a proprietary oil-in- water emulsion based Adjuvant System proven to be well-tolerated and highly immunogenic in the human (Leroux-Roels et al. (2007) The Lancet 370:580–589), for its ability to induce intra-subtypic cross-protection against clade 2 H5N1/A/Indonesia/5/05 challenge in ferrets.

Methodology and Principal Findings

All ferrets in control groups receiving non-adjuvanted vaccine or adjuvant alone failed to develop specific or cross-reactive neutralizing antibodies and all died or had to be euthanized within four days of virus challenge. Two doses of adjuvanted split H5N1 vaccine containing ≥1.7 µg HA induced neutralizing antibodies in the majority of ferrets to both clade 1 (17/23 (74%) responders) and clade 2 viruses (14/23 (61%) responders), and 96% (22/23) of vaccinees survived the lethal challenge. Furthermore lung virus loads and viral shedding in the upper respiratory tract were reduced in vaccinated animals relative to controls suggesting that vaccination might also confer a reduced risk of viral transmission.

Conclusion

These protection data in a stringent challenge model in association with an excellent clinical profile highlight the potential of this adjuvanted H5N1 candidate vaccine as an effective tool in pandemic preparedness.     

Vaccination against seasonal influenza A/H3N2 virus reduces the induction of heterosubtypic immunity against influenza A/H5N1 virus infection in ferrets

Infection with seasonal influenza viruses induces a certain extent of protective immunity against potentially pandemic viruses of novel subtypes, also known as heterosubtypic immunity. Here we demonstrate that infection with a recent influenza A/H3N2 virus strain induces robust protection in ferrets against infection with a highly pathogenic avian influenza virus of the H5N1 subtype. Prior H3N2 virus infection reduced H5N1 virus replication in the upper respiratory tract, as well as clinical signs, mortality, and histopathological changes associated with virus replication in the brain. This protective immunity correlated with the induction of T cells that cross-reacted with H5N1 viral antigen. We also demonstrated that prior vaccination against influenza A/H3N2 virus reduced the induction of heterosubtypic immunity otherwise induced by infection with the influenza A/H3N2 virus. The implications of these findings are discussed in the context of vaccination strategies and vaccine development aiming at the induction of immunity to pandemic influenza.     

Generation of High-yield Vaccine Strain Wholly Derived from Avian Influenza Viruses by Reverse Genetics

Highly pathogenic avian influenza A (HPAI) viruses of the H5N1 subtypes caused enormous economical loss to poultry farms in China and Southeastern Asian countries. The vaccination program is a reliable strategy in controlling the prevalence of these disastrous diseases. The six internal genes of the high-yield influenza virus A/Goose/Dalian/3/01 (H9N2), the haemagglutinin (HA) gene of A/Goose/HLJ/QFY/04 (H5N1) strain, and the neuraminidase gene from A/Duck/Germany/1215/73 (H2N3) reference strain were amplified by RT-PCR technique. The HA gene was modified by the deletion of four basic amino acids of the connecting peptide between HA1 and HA2. Eight gene expressing plasmids were constructed, and the recombinant virus rH5N3 were generated by cell transfection. The infection of chicken embryos and the challenge tests involving chickens demonstrated that the recombinant H5N3 (rH5N3) influenza virus is avirulent. The allantoic fluids of rH5N3-infected eggs contain high-titer influenza viruses with haemagglutination unit of 1:2 048, which are eight times those of the parental H5N1 virus. The rH5N3 oil-emulsified vaccine could induce haemagglutination inhibition (HI) antibodies in chickens in 2 weeks post-vaccination, and the maximum geometric mean HI-titers were observed 4–5 weeks post-vaccination and were kept under observation for 18 weeks. The rH5N3-vaccinated chickens were fully protected against morbidity and mortality of the lethal challenge of the H5N1 HPAI viruses, A/Goose/Guangdong/1/96 and A/Goose/HLJ/QFY/04, which had 8 years expansion and differences among multiple amino acids in HA protein. The N3 neuraminidase protein marker makes it possible to distinguish between H5N1-infected and H5N3-vaccinated animals.     

Vaccine-Induced Boosting of Influenza Virus-Specific CD4 T Cells in Younger and Aged Humans

Current yearly influenza virus vaccines induce strain-specific neutralizing antibody (NAb) responses providing protective immunity to closely matched viruses. However, these vaccines are often poorly effective in high-risk groups such as the elderly and challenges exist in predicting yearly or emerging pandemic influenza virus strains to include in the vaccines. Thus, there has been considerable emphasis on understanding broadly protective immunological mechanisms for influenza virus. Recent studies have implicated memory CD4 T cells in heterotypic immunity in animal models and in human challenge studies. Here we examined how influenza virus vaccination boosted CD4 T cell responses in younger versus aged humans. Our results demonstrate that while the magnitude of the vaccine-induced CD4 T cell response and number of subjects responding on day 7 did not differ between younger and aged subjects, fewer aged subjects had peak responses on day 14. While CD4 T cell responses were inefficiently boosted against NA, both HA and especially nucleocaspid protein- and matrix-(NP+M) specific responses were robustly boosted. Pre-existing CD4 T cell responses were associated with more robust responses to influenza virus NP+M, but not H1 or H3. Finally pre-existing strain-specific NAb decreased the boosting of CD4 T cell responses. Thus, accumulation of pre-existing influenza virus-specific immunity in the form of NAb and cross-reactive T cells to conserved virus proteins (e.g. NP and M) over a lifetime of exposure to infection and vaccination may influence vaccine-induced CD4 T cell responses in the aged.     

Protection against a Lethal Avian Influenza A Virus in a Mammalian System

The question of how best to protect the human population against a potential influenza pandemic has been raised by the recent outbreak caused by an avian H5N1 virus in Hong Kong. The likely strategy would be to vaccinate with a less virulent, laboratory-adapted H5N1 strain isolated previously from birds. Little attention has been given, however, to dissecting the consequences of sequential exposure to serologically related influenza A viruses using contemporary immunology techniques. Such experiments with the H5N1 viruses are limited by the potential risk to humans. An extremely virulent H3N8 avian influenza A virus has been used to infect both immunoglobulin-expressing (Ig^+/+) and Ig^−/− mice primed previously with a laboratory-adapted H3N2 virus. The cross-reactive antibody response was very protective, while the recall of CD8⁺ T-cell memory in the Ig^−/− mice provided some small measure of resistance to a low-dose H3N8 challenge. The H3N8 virus also replicated in the respiratory tracts of the H3N2-primed Ig^+/+ mice, generating secondary CD8⁺ and CD4⁺ T-cell responses that may contribute to recovery. The results indicate that the various components of immune memory operate together to provide optimal protection, and they support the idea that related viruses of nonhuman origin can be used as vaccines.