Gastrointestinal Delivery of Baculovirus Displaying Influenza Virus Hemagglutinin Protects Mice against Heterologous H5N1 Infection

The recent outbreaks of influenza A H5N1 virus in birds and humans have necessitated the development of potent H5N1 vaccines. In this study, we evaluated the protective potential of an immediate-early promoter-based baculovirus displaying hemagglutinin (BacHA) against highly pathogenic avian influenza (HPAI) H5N1 virus infection in a mouse model. Gastrointestinal delivery of BacHA significantly enhanced the systemic immune response in terms of HA-specific serum IgG and hemagglutination inhibition (HI) titers. In addition, BacHA vaccine was able to significantly enhance the mucosal IgA level. The inclusion of recombinant cholera toxin B subunit as a mucosal adjuvant along with BacHA vaccine did not influence either the systemic or mucosal immunity. Interestingly, an inactivated form of BacHA was able to induce only a negligible level of immune responses compared to its live counterpart. Microneutralization assay also indicated that live BacHA vaccine was able to induce strong cross-clade neutralization against heterologous H5N1 strains (clade 1.0, clade 2.1, and clade 8.0) compared to the inactivated BacHA. Viral challenge studies showed that live BacHA was able to provide 100% protection against 5 50% mouse lethal doses (MLD₅₀) of homologous (clade 2.1) and heterologous (clade 1) H5N1. Moreover, histopathological examinations revealed that mice vaccinated with live BacHA had only minimal bronchitis in lungs and regained their body weight more rapidly postchallenge. Furthermore, immunohistochemistry results demonstrated that the live BacHA was able to transduce and express HA in the intestinal epithelial cells in vitro and in vivo. We have demonstrated that recombinant baculovirus with a white spot syndrome virus (WSSV) immediate-early promoter 1 (ie1) acted as a vector as well as a protein vaccine and will enable the rapid production of prepandemic and pandemic vaccines without any biosafety concerns.The recent outbreaks of H5N1 avian flu and the current pandemic situation with H1N1 swine-origin influenza A virus (S-OIV) are clear indications of the urgent need for effective vaccines against influenza A viruses (31). Preventive and therapeutic measures against influenza A viruses have received much interest and effort globally to combat the current pandemic and to prevent such a situation in the future. Currently used vaccines for influenza are administered mainly parenterally and include live attenuated reassortant viruses, conventional inactivated whole viral antigens, or split-virus vaccines. Although some of these vaccines have proven to be quite effective, the manufacturing of these vaccines involves several technical and safety issues (21). Furthermore, the production of currently available influenza vaccines often requires high-level biocontainment facilities, an additional hurdle that limits the advancement of present vaccines.Vaccines containing purified recombinant viral proteins have recently gained special attention due to their ease of production without any safety concerns (25). Recombinant hemagglutinin (rHA) subunit vaccines produced in baculovirus-insect cell expression systems have been extensively tested and evaluated in humans (29, 30). Baculovirus-derived rHA subunit vaccines administered parenterally are safe and immunogenic in animals and humans. Along with its success in recombinant protein vaccines, baculovirus surface display technology allows us to present large complex proteins on the baculovirus envelope in its native antigenic conformation, resulting in good stability and a longer half-life in the host (18, 14, 8).Along with a suitable antigen, the route of administration of the vaccine has a profound effect in controlling mucosally acquired infections such as influenza. Vaccination via the mucosal route stimulates both systemic and mucosal immune responses (16). Oral and intranasal vaccines are the two main options for mucosal administration. Intranasal vaccines would have a detrimental effect on persons with asthma, reactive airway disease, and other chronic pulmonary or cardiovascular disorders (4). Oral vaccines therefore seem to be the safest alternative (13). Moreover, there is evidence to prove the ability of oral vaccination to prevent infection of the lungs (23) and cause transcytosis of the molecule across the cells into the circulation (24).In this report, we describe the construction of recombinant baculovirus under control of the immediate-early promoter 1 (ie1) derived from the white spot syndrome virus (WSSV) genome, which enables the expression of hemagglutinin at the early stage of infection in insect cells, thereby enhancing the display of HA on the baculovirus envelope. Incorporation of more HA into the budding baculovirus particles would improve their efficacy as immunogens. We have studied the efficacy of WSSV ie1-based baculovirus displaying hemagglutinin (BacHA) as an oral vaccine in a mouse model of infection. We have also assessed its efficacy with recombinant cholera toxin B (rCTB) as a mucosal adjuvant. This strategy will enable rapid production of prepandemic vaccines with minimal infrastructure around the world, alleviating the need for high-biosafety facilities, risky inactivation of virulent viruses, and meticulous protein purification procedures.     

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.     

A Virus-Like Particle That Elicits Cross-Reactive Antibodies to the Conserved Stem of Influenza Virus Hemagglutinin

The discovery of broadly neutralizing antibodies that recognize highly conserved epitopes in the membrane-proximal region of influenza virus hemagglutinin (HA) has revitalized efforts to develop a universal influenza virus vaccine. This effort will likely require novel immunogens that contain these epitopes but lack the variable and immunodominant epitopes located in the globular head of HA. As a first step toward developing such an immunogen, we investigated whether the 20-residue A-helix of the HA2 chain that forms the major component of the epitope of broadly neutralizing antibodies CR6261, F10, and others is sufficient by itself to elicit antibodies with similarly broad antiviral activity. Here, we report the multivalent display of the A-helix on icosahedral virus-like particles (VLPs) derived from the capsid of Flock House virus. Mice immunized with VLPs displaying 180 copies/particle of the A-helix produced antibodies that recognized trimeric HA and the elicited antibodies had binding characteristics similar to those of CR6261 and F10: they recognized multiple HA subtypes from group 1 but not from group 2. However, the anti-A-helix antibodies did not neutralize influenza virus. These results indicate that further engineering of the transplanted peptide is required and that display of additional regions of the epitope may be necessary to achieve protection.     

DNA Vaccine Encoding Hemagglutinin Provides Protective Immunity against H5N1 Influenza Virus Infection in Mice

In Hong Kong in 1997, a highly lethal H5N1 avian influenza virus was apparently transmitted directly from chickens to humans with no intermediate mammalian host and caused 18 confirmed infections and six deaths. Strategies must be developed to deal with this virus if it should reappear, and prospective vaccines must be developed to anticipate a future pandemic. We have determined that unadapted H5N1 viruses are pathogenic in mice, which provides a well-defined mammalian system for immunological studies of lethal avian influenza virus infection. We report that a DNA vaccine encoding hemagglutinin from the index human influenza isolate A/HK/156/97 provides immunity against H5N1 infection of mice. This immunity was induced against both the homologous A/HK/156/97 (H5N1) virus, which has no glycosylation site at residue 154, and chicken isolate A/Ck/HK/258/97 (H5N1), which does have a glycosylation site at residue 154. The mouse model system should allow rapid evaluation of the vaccine’s protective efficacy in a mammalian host. In our previous study using an avian model, DNA encoding hemagglutinin conferred protection against challenge with antigenic variants that differed from the primary antigen by 11 to 13% in the HA1 region. However, in our current study we found that a DNA vaccine encoding the hemagglutinin from A/Ty/Ir/1/83 (H5N8), which differs from A/HK/156/97 (H5N1) by 12% in HA1, prevented death but not H5N1 infection in mice. Therefore, a DNA vaccine made with a heterologous H5 strain did not prevent infection by H5N1 avian influenza viruses in mice but was useful in preventing death.     

Nanodisc-Incorporated Hemagglutinin Provides Protective Immunity against Influenza Virus Infection

Every year, influenza virus infection causes significant mortality and morbidity in human populations. Although egg-based inactivated viral vaccines are available, their effectiveness depends on the correct prediction of the circulating viral strains and is limited by the time constraint of the manufacturing process. Recombinant subunit vaccines are easier to manufacture with a relatively short lead time but are limited in their efficacy partly because the purified recombinant membrane proteins in the soluble form most likely do not retain their native membrane-bound structure. Nanodisc (ND) particles are soluble, stable, and reproducibly prepared discoid shaped nanoscale structures that contain a discrete lipid bilayer bound by two amphipathic scaffold proteins. Because ND particles permit the functional reconstitution of membrane/envelope proteins, we incorporated recombinant hemagglutinin (HA) from influenza virus strain A/New Caledonia/20/99 (H1N1) into NDs and investigated their potential to elicit an immune response to HA and confer immunity to influenza virus challenge relative to the commercial vaccines Fluzone and FluMist. HA-ND vaccination induced a robust anti-HA antibody response consisting of predominantly the immunoglobulin G1 (IgG1) subclass and a high hemagglutination inhibition titer. Intranasal immunization with HA-ND induced an anti-HA IgA response in nasal passages. HA-ND vaccination conferred protection that was comparable to that of Fluzone and FluMist against challenge with influenza virus strain A/Puerto Rico/8/1934 (H1N1).The influenza A virus-type viral genome encodes 11 proteins including hemagglutinin (HA) and neuraminidase (NA). HA is important in virus transmission and is also a major determinant of host range (16). NA prevents viral aggregation and helps in the release of new viruses from the infected cell (25). These glycoproteins are the principal antigens against which humoral immune responses of the host are directed. Vaccination has been accepted as the most effective method of preventing influenza virus. Current licensed vaccines against influenza virus include conventional inactivated virus vaccine, live-attenuated vaccine, or inactivated “split-virus” vaccines, all grown in embryonated chicken eggs. Influenza virus vaccines may contain residual egg-derived antigens, which is a risk factor for persons with hypersensitivity to eggs. In the case of live-attenuated vaccines that are delivered by the mucosal route, there are several potential safety concerns including the possibility that the vaccine strain could undergo spontaneous genetic change and in a rare case of simultaneous infection with another influenza virus could undergo antigenic shift. These factors are of special concern for children and the elderly, who are the primary populations at risk for influenza virus infection (9). Therefore, there is a continuing need for developing more efficacious and safer vaccines.Apart from licensed vaccines, a number of different vaccine formulations including soluble glycoproteins, virus-like particles, and subunit vaccines (6, 9, 14) with various efficacies have been developed. Recombinant glycoprotein vaccines offer many distinct advantages, including cost, the possibility of adapting them to rapidly changing strains within a short time, and independence from egg-based formulations. In experimental setups, recombinant HA (rHA) and recombinant NA have provided protection against lethal challenge to mice (18, 27). The safety, immunogenicity, and efficacy of trivalent rHA vaccines have been established (26), and a potential trivalent HA vaccine (FluBlok; Protein Sciences Corporation) is currently in phase III clinical trials.Some rHA-based vaccines elicit high titers of anti-HA antibodies. However, these antibodies do not necessarily possess a high capacity for virus neutralization. This apparent discrepancy likely results from the use of soluble HA protein that may not accurately mimic the native structure of the membrane-embedded glycoprotein on the viral envelope for immunization. This could result in a robust antibody response with a limited ability to react with “native epitopes.” This notion is supported by data from previously reported studies that indicated that antigens expressed in their native three-dimensional conformation can elicit a more effective antibody response than proteins in their nonnative forms (19). Therefore, we investigated whether rHA presented in a lipid-bilayer-embedded formulation would elicit a potent neutralizing antibody response.The Nanodisc (ND) system was developed as a novel method for functionally reconstituting membrane proteins into soluble nanoscale lipid bilayers (3, 4, 12, 22). NDs are robust, reproducible, and monodisperse discoidal particles 5.5 nm high and nominally 10 nm in diameter that are formed via a self-assembly process. ND particles contain two copies of an alpha-helical, amphipathic protein, termed membrane scaffold protein (MSP), which encircles a lipid bilayer in a “belt-like” fashion (Fig. ​(Fig.1a).1a). A mixture of phospholipids and MSP are placed in a nonequilibrium solubilized state, for instance, using detergent or high hydrostatic pressure, and the system is then allowed to approach equilibrium by the gentle removal of the perturbant. This initiates a process of self-assembly, wherein the phospholipids and MSP find each other and generate a discoidal phospholipid bilayer encircled by the MSP. The resulting nanostructures represent a highly stable and homogeneous population with an aqueous solubility in the millimolar range (11).Open in a separate windowFIG. 1.Construction of HA-NDs. (a) Schematic showing an ND particle that contains a phospholipid bilayer encircled by membrane scaffold proteins (left) (5) and the same ND particle with an embedded transmembrane protein (right) (17). (b) HA-ND assemblies were first purified by Ni²⁺ affinity chromatography. (Top left) Silver-stained SDS-PAGE showing flowthrough, wash, and elution of HA-ND assembly mix over a Ni-nitrilotriacetic acid column (FT1 and FT2 are flowthrough, and the eluate contains the eluted protein). Arrows show the positions of the 72-kDa HA band and the 25-kDa MSPs. (Top right) Anti-HA Western blotting of the same SDS-PAGE gel. Depending on the quality of purification, a certain fraction of full-length 72-kDa rHA (HA0) can exist as proteolytically cleaved HA1 (∼50-kDa) and HA2 (∼28-kDa) subunits. (Bottom left) Ni²⁺ column eluates were further purified by SEC. Silver-stained SDS-PAGE gel shows size-based fractionation of Ni²⁺ column eluate. The numbers at the bottom correspond to the fractions collected. The MSP amounts are largest at fractions 27 to 30, showing that empty NDs eluted at those fractions. (Bottom right) Anti-HA Western blotting of the same SDS-PAGE gel showing that HA-ND assemblies eluted mainly between fractions 18 and 26. (c) Elution profile of HA-ND following SEC separation. The elution times for protein standards used for calibration are indicated at the top. The control profile for empty NDs is superimposed. HA-ND assemblies have a shorter retention time than empty NDs. inj, injection. (d) HA-ND assemblies from different SEC fractions separate as discrete-sized molecules upon native PAGE separation. Silver staining (left) and anti-HA Western blotting (right) of native PAGE gels from size exclusion fractions show different HA polymers contained in NDs. Earlier fractions are rich in higher-polymeric forms of HA, while later fractions are richer in monomeric HA. Control HA was loaded in the last well to the right in both cases.The value of the ND self-assembly process is that one can simply and reproducibly incorporate membrane proteins into these structures. This is accomplished by including the membrane protein in the initial mixture of MSP, lipid, and detergent prior to the initiation of the self-assembly process. An incorporated membrane protein then finds itself in a native-like environment with stability and activity normally found in vivo. By using phospholipids with different chemical characteristics (charge, degree of unsaturation, and length of acyl chains), the bilayer environment can be optimized to accommodate functional requirements. Furthermore, larger scaffold proteins, which in turn create a larger-diameter particle, can be employed to incorporate multimers or membrane protein complexes. Numerous membrane proteins from the three major classes-integral, tethered, and embedded (including monomers and multimers)-in the lipid bilayer environment created by NDs have been studied (2-5, 8, 10, 13, 20, 23). Since the ND system creates a stable bilayer environment that mimics that encountered by a membrane protein in the cell membrane, membrane proteins display normal folding, native ligand binding kinetics, and intact signaling activity (1, 3, 5, 8, 10, 13, 17, 23).In this study, we successfully incorporated recombinant baculovirus-derived HA into NDs (HA-ND) and compared its efficacy to induce a relevant immune response and confer protection against influenza virus challenge with those of existing licensed vaccines by using a mouse model.     

Intranasal Immunization of Baculovirus Displayed Hemagglutinin Confers Complete Protection against Mouse Adapted Highly Pathogenic H7N7 Reassortant Influenza Virus

Background

Avian influenza A H7N7 virus poses a pandemic threat to human health because of its ability for direct transmission from domestic poultry to humans and from human to human. The wide zoonotic potential of H7N7 combined with an antiviral immunity inhibition similar to pandemic 1918 H1N1 and 2009 H1N1 influenza viruses is disconcerting and increases the risk of a putative H7N7 pandemic in the future, underlining the urgent need for vaccine development against this virus.

Methodology/Principal Findings

In this study, we developed a recombinant vaccine by expressing the H7N7-HA protein on the surface of baculovirus (Bac-HA). The protective efficacy of the live Bac-HA vaccine construct was evaluated in a mouse model by challenging mice immunized intranasally (i.n.) or subcutaneously (s.c.) with high pathogenic mouse adapted H7N7 reassorted strain. Although s.c. injection of live Bac-HA induced higher specific IgG than i.n. immunization, the later resulted in an elevated neutralization titer. Interestingly, 100% protection from the lethal viral challenge was only observed for the mice immunized intranasally with live Bac-HA, whereas no protection was achieved in any other s.c. or i.n. immunized mice groups. In addition, we also observed higher mucosal IgA as well as increased IFN-γ and IL-4 responses in the splenocytes of the surviving mice coupled with a reduced viral titer and diminished histopathological signs in the lungs.

Conclusion

Our results indicated that protection from high pathogenic H7N7 (NL/219/03) virus requires both mucosal and systemic immune responses in mice. The balance between Th1 and Th2 cytokines is also required for the protection against the H7N7 pathogen. Intranasal administration of live Bac-HA induced all these immune responses and protected the mice from lethal viral challenge. Therefore, live Bac-HA is an effective vaccine candidate against H7N7 viral infections.     

Intranasal Immunization with Pressure Inactivated Avian Influenza Elicits Cellular and Humoral Responses in Mice

Influenza viruses pose a serious global health threat, particularly in light of newly emerging strains, such as the avian influenza H5N1 and H7N9 viruses. Vaccination remains the primary method for preventing acquiring influenza or for avoiding developing serious complications related to the disease. Vaccinations based on inactivated split virus vaccines or on chemically inactivated whole virus have some important drawbacks, including changes in the immunogenic properties of the virus. To induce a greater mucosal immune response, intranasally administered vaccines are highly desired as they not only prevent disease but can also block the infection at its primary site. To avoid these drawbacks, hydrostatic pressure has been used as a potential method for viral inactivation and vaccine production. In this study, we show that hydrostatic pressure inactivates the avian influenza A H3N8 virus, while still maintaining hemagglutinin and neuraminidase functionalities. Challenged vaccinated animals showed no disease signs (ruffled fur, lethargy, weight loss, and huddling). Similarly, these animals showed less Evans Blue dye leakage and lower cell counts in their bronchoalveolar lavage fluid compared with the challenged non-vaccinated group. We found that the whole inactivated particles were capable of generating a neutralizing antibody response in serum, and IgA was also found in nasal mucosa and feces. After the vaccination and challenge we observed Th1/Th2 cytokine secretion with a prevalence of IFN-γ. Our data indicate that the animals present a satisfactory immune response after vaccination and are protected against infection. Our results may pave the way for the development of a novel pressure-based vaccine against influenza virus.     

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.     

Protective Efficacy of Newcastle Disease Virus Expressing Soluble Trimeric Hemagglutinin against Highly Pathogenic H5N1 Influenza in Chickens and Mice

Background

Highly pathogenic avian influenza virus (HPAIV) causes a highly contagious often fatal disease in poultry, resulting in significant economic losses in the poultry industry. HPAIV H5N1 also poses a major public health threat as it can be transmitted directly from infected poultry to humans. One effective way to combat avian influenza with pandemic potential is through the vaccination of poultry. Several live vaccines based on attenuated Newcastle disease virus (NDV) that express influenza hemagglutinin (HA) have been developed to protect chickens or mammalian species against HPAIV. However, the zoonotic potential of NDV raises safety concerns regarding the use of live NDV recombinants, as the incorporation of a heterologous attachment protein may result in the generation of NDV with altered tropism and/or pathogenicity.

Methodology/Principal Findings

In the present study we generated recombinant NDVs expressing either full length, membrane-anchored HA of the H5 subtype (NDV-H5) or a soluble trimeric form thereof (NDV-sH5³). A single intramuscular immunization with NDV-sH5³ or NDV-H5 fully protected chickens against disease after a lethal challenge with H5N1 and reduced levels of virus shedding in tracheal and cloacal swabs. NDV-sH5³ was less protective than NDV-H5 (50% vs 80% protection) when administered via the respiratory tract. The NDV-sH5³ was ineffective in mice, regardless of whether administered oculonasally or intramuscularly. In this species, NDV-H5 induced protective immunity against HPAIV H5N1, but only after oculonasal administration, despite the poor H5-specific serum antibody response it elicited.

Conclusions/Significance

Although NDV expressing membrane anchored H5 in general provided better protection than its counterpart expressing soluble H5, chickens could be fully protected against a lethal challenge with H5N1 by using the latter NDV vector. This study thus provides proof of concept for the use of recombinant vector vaccines expressing a soluble form of a heterologous viral membrane protein. Such vectors may be advantageous as they preclude the incorporation of heterologous membrane proteins into the viral vector particles.     

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 AlPO₄ 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.     

Hemagglutinin Stalk-Based Universal Vaccine Constructs Protect against Group 2 Influenza A Viruses

Current influenza virus vaccines contain H1N1 (phylogenetic group 1 hemagglutinin), H3N2 (phylogenetic group 2 hemagglutinin), and influenza B virus components. These vaccines induce good protection against closely matched strains by predominantly eliciting antibodies against the membrane distal globular head domain of their respective viral hemagglutinins. This domain, however, undergoes rapid antigenic drift, allowing the virus to escape neutralizing antibody responses. The membrane proximal stalk domain of the hemagglutinin is much more conserved compared to the head domain. In recent years, a growing collection of antibodies that neutralize a broad range of influenza virus strains and subtypes by binding to this domain has been isolated. Here, we demonstrate that a vaccination strategy based on the stalk domain of the H3 hemagglutinin (group 2) induces in mice broadly neutralizing anti-stalk antibodies that are highly cross-reactive to heterologous H3, H10, H14, H15, and H7 (derived from the novel Chinese H7N9 virus) hemagglutinins. Furthermore, we demonstrate that these antibodies confer broad protection against influenza viruses expressing various group 2 hemagglutinins, including an H7 subtype. Through passive transfer experiments, we show that the protection is mediated mainly by neutralizing antibodies against the stalk domain. Our data suggest that, in mice, a vaccine strategy based on the hemagglutinin stalk domain can protect against viruses expressing divergent group 2 hemagglutinins.     

Microneedle Vaccination Elicits Superior Protection and Antibody Response over Intranasal Vaccination against Swine-Origin Influenza A (H1N1) in Mice

Influenza is one of the critical infectious diseases globally and vaccination has been considered as the best way to prevent. In this study, immunogenicity and protection efficacy between intranasal (IN) and microneedle (MN) vaccination was compared using inactivated swine-origin influenza A/H1N1 virus vaccine. Mice were vaccinated by MN or IN administration with 1 μg of inactivated H1N1 virus vaccine. Antigen-specific antibody responses and hemagglutination-inhibition (HI) titers were measured in all immunized sera after immunization. Five weeks after an immunization, a lethal challenge was performed to evaluate the protective efficacy. Furthermore, mice were vaccinated by IN administration with higher dosages (> 1 μg), analyzed in the same manner, and compared with 1 μg-vaccine-coated MN. Significantly higher antigen-specific antibody responses and HI titer were measured in sera in MN group than those in IN group. While 100% protection, slight weight loss, and reduced viral replication were observed in MN group, 0% survival rate were observed in IN group. As vaccine dose for IN vaccination increased, MN-immunized sera showed much higher antigen-specific antibody responses and HI titer than other IN groups. In addition, protective immunity of 1 μg-MN group was similar to those of 20- and 40 μg-IN groups. We conclude that MN vaccination showed more potential immune response and protection than IN vaccination at the same vaccine dosage.     

Improving Cross-Protection against Influenza Virus Using Recombinant Vaccinia Vaccine Expressing NP and M2 Ectodomain Tandem Repeats

Conventional influenza vaccines need to be designed and manufactured yearly. However, they occasionally provide poor protection owing to antigenic mismatch. Hence, there is an urgent need to develop universal vaccines against influenza virus. Using nucleoprotein(NP) and extracellular domain of matrix protein 2(M2e) genes from the influenza A virus A/Beijing/30/95(H3N2), we constructed four recombinant vaccinia virus-based influenza vaccines carrying NP fused with one or four copies of M2e genes in different orders. The recombinant vaccinia viruses were used to immunize BALB/C mice. Humoral and cellular responses were measured, and then the immunized mice were challenged with the influenza A virus A/Puerto Rico/8/34(PR8). NP-specific humoral response was elicited in mice immunized with recombinant vaccinia viruses carrying full-length NP, while robust M2e-specific humoral response was elicited only in the mice immunized with recombinant vaccinia viruses carrying multiple copies of M2e. All recombinant viruses elicited NP-and M2e-specific cellular immune responses in mice. Only immunization with RVJ-4M2eNP induced remarkably higher levels of IL-2 and IL-10 cytokines specific to M2e. Furthermore, RVJ-4M2eNP immunization provided the highest cross-protection in mice challenged with 20 MLD_(50) of PR8. Therefore, the cross-protection potentially correlates with both NP and M2e-specific humoral and cellular immune responses induced by RVJ-4M2eNP, which expresses a fusion antigen of full-length NP preceded by four M2e repeats. These results suggest that the rational fusion of NP and multiple M2e antigens is critical toward inducing protective immune responses, and the 4M2eNP fusion antigen may be employed to develop a universal influenza vaccine.     

Coadministration of DNA Encoding Interleukin-6 and Hemagglutinin Confers Protection from Influenza Virus Challenge in Mice

This study was conducted to investigate whether Accell gene gun coadministration of DNA encoding human interleukin-6 (IL-6) would enhance protective immune responses in mice to an equine influenza A virus hemagglutinin (HA) DNA vaccine. Mice that received HA DNA alone exhibited accelerated clearance of homologous challenge virus but were not protected from infection. In contrast, mice that received both HA and IL-6 DNA had no detectable virus in their lungs after challenge. These results strongly support the use of IL-6 as a cytokine adjuvant in DNA vaccination.     

Molecular Signatures of Hemagglutinin Stem-Directed Heterosubtypic Human Neutralizing Antibodies against Influenza A Viruses

Recent studies have shown high usage of the IGHV1-69 germline immunoglobulin gene for influenza hemagglutinin stem-directed broadly-neutralizing antibodies (HV1-69-sBnAbs). Here we show that a major structural solution for these HV1-69-sBnAbs is achieved through a critical triad comprising two CDR-H2 loop anchor residues (a hydrophobic residue at position 53 (Ile or Met) and Phe54), and CDR-H3-Tyr at positions 98±1; together with distinctive V-segment CDR amino acid substitutions that occur in positions sparse in AID/polymerase-η recognition motifs. A semi-synthetic IGHV1-69 phage-display library screen designed to investigate AID/polη restrictions resulted in the isolation of HV1-69-sBnAbs that featured a distinctive Ile52Ser mutation in the CDR-H2 loop, a universal CDR-H3 Tyr at position 98 or 99, and required as little as two additional substitutions for heterosubtypic neutralizing activity. The functional importance of the Ile52Ser mutation was confirmed by mutagenesis and by BCR studies. Structural modeling suggests that substitution of a small amino acid at position 52 (or 52a) facilitates the insertion of CDR-H2 Phe54 and CDR-H3-Tyr into adjacent pockets on the stem. These results support the concept that activation and expansion of a defined subset of IGHV1-69-encoded B cells to produce potent HV1-69-sBnAbs does not necessarily require a heavily diversified V-segment acquired through recycling/reentry into the germinal center; rather, the incorporation of distinctive amino acid substitutions by Phase 2 long-patch error-prone repair of AID-induced mutations or by random non-AID SHM events may be sufficient. We propose that these routes of B cell maturation should be further investigated and exploited as a pathway for HV1-69-sBnAb elicitation by vaccination.     

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.