Influenza virus-like particles containing M2 induce broadly cross protective immunity

Background

Current influenza vaccines based on the hemagglutinin protein are strain specific and do not provide good protection against drifted viruses or emergence of new pandemic strains. An influenza vaccine that can confer cross-protection against antigenically different influenza A strains is highly desirable for improving public health.

Methodology/Principal Findings

To develop a cross protective vaccine, we generated influenza virus-like particles containing the highly conserved M2 protein in a membrane-anchored form (M2 VLPs), and investigated their immunogenicity and breadth of cross protection. Immunization of mice with M2 VLPs induced anti-M2 antibodies binding to virions of various strains, M2 specific T cell responses, and conferred long-lasting cross protection against heterologous and heterosubtypic influenza viruses. M2 immune sera were found to play an important role in providing cross protection against heterosubtypic virus and an antigenically distinct 2009 pandemic H1N1 virus, and depletion of dendritic and macrophage cells abolished this cross protection, providing new insight into cross-protective immune mechanisms.

Conclusions/Significance

These results suggest that presenting M2 on VLPs in a membrane-anchored form is a promising approach for developing broadly cross protective influenza vaccines.     

T- and B-Cell-Mediated Protection Induced by Novel,Live Attenuated Pertussis Vaccine in Mice. Cross Protection against Parapertussis

Background

Despite the extensive use of efficacious vaccines, pertussis still ranks among the major causes of childhood mortality worldwide. Two types of pertussis vaccines are currently available, whole-cell, and the more recent acellular vaccines. Because of reduced reactogenicity and comparable efficacy acellular vaccines progressively replace whole-cell vaccines. However, both types require repeated administrations for optimal efficacy. We have recently developed a live attenuated vaccine candidate, named BPZE1, able to protect infant mice after a single nasal administration.

Methodology/Principal Findings

We determined the protective mechanism of BPZE1-mediated immunity by using passive transfer of T cells and antibodies from BPZE1-immunized mice to SCID mice. Clearance of Bordetella pertussis from the lungs was mediated by both BPZE1-induced antibodies and CD4⁺, but not by CD8⁺ T cells. The protective CD4⁺ T cells comprised IFN-γ-producing and IL-17-producing subsets, indicating that BPZE1 induces both Th1 and Th17 CD4⁺ T cells. In addition, and in contrast to acellular pertussis vaccines, BPZE1 also cross-protected against Bordetella parapertussis infection, but in this case only the transfer of CD4⁺ T cells conferred protection. Serum from BPZE1-immunized mice was not able to kill B. parapertussis and did not protect SCID mice against B. parapertussis infection.

Conclusions/Significance

The novel live attenuated pertussis vaccine BPZE1 protects in a pre-clinical mouse model against B. pertussis challenge by both BPZE1-induced antibodies and CD4⁺ T cell responses. It also protects against B. parapertussis infection. However, in this case protection is only T cell mediated.     

Cross-clade protective immune responses to influenza viruses with H5N1 HA and NA elicited by an influenza virus-like particle

Background

Vaccination is a cost-effective counter-measure to the threat of seasonal or pandemic outbreaks of influenza. To address the need for improved influenza vaccines and alternatives to egg-based manufacturing, we have engineered an influenza virus-like particle (VLP) as a new generation of non-egg or non-mammalian cell culture-based candidate vaccine.

Methodology/Principal Findings

We generated from a baculovirus expression system using insect cells, a non-infectious recombinant VLP vaccine from both influenza A H5N1 clade 1 and clade 2 isolates with pandemic potential. VLPs were administered to mice in either a one-dose or two-dose regimen and the immune responses were compared to those induced by recombinant hemagglutinin (rHA). Both humoral and cellular responses were analyzed. Mice vaccinated with VLPs were protected against challenge with lethal reassortant viruses expressing the H5N1 HA and NA, regardless if the H5N1 clade was homologous or heterologous to the vaccine. However, rHA-vaccinated mice showed considerable weight loss and death following challenge with the heterovariant clade virus. Protection against death induced by VLPs was independent of the pre-challenge HAI titer or cell-mediated responses to HA or M1 since vaccinated mice, with low to undetectable cross-clade HAI antibodies or cellular responses to influenza antigens, were still protected from a lethal viral challenge. However, an apparent association rate of antibody binding to HA correlated with protection and was enhanced using VLPs, particularly when delivered intranasally, compared to rHA vaccines.

Conclusion/Significance

This is the first report describing the use of an H5N1 VLP vaccine created from a clade 2 isolate. The results show that a non-replicating virus-like particle is effective at eliciting a broadened, cross-clade protective immune response to proteins from emerging H5N1 influenza isolates giving rise to a potential pandemic influenza vaccine candidate for humans that can be stockpiled for use in the event of an outbreak of H5N1 influenza.     

Single-dose mucosal immunization with a candidate universal influenza vaccine provides rapid protection from virulent H5N1, H3N2 and H1N1 viruses

Background

The sudden emergence of novel influenza viruses is a global public health concern. Conventional influenza vaccines targeting the highly variable surface glycoproteins hemagglutinin and neuraminidase must antigenically match the emerging strain to be effective. In contrast, “universal” vaccines targeting conserved viral components could be used regardless of viral strain or subtype. Previous approaches to universal vaccination have required protracted multi-dose immunizations. Here we evaluate a single dose universal vaccine strategy using recombinant adenoviruses (rAd) expressing the conserved influenza virus antigens matrix 2 and nucleoprotein.

Methodology/Principal Findings

In BALB/c mice, administration of rAd via the intranasal route was superior to intramuscular immunization for induction of mucosal responses and for protection against highly virulent H1N1, H3N2, or H5N1 influenza virus challenge. Mucosally vaccinated mice not only survived, but had little morbidity and reduced lung virus titers. Protection was observed as early as 2 weeks post-immunization, and lasted at least 10 months, as did antibodies and lung T cells with activated phenotypes. Virus-specific IgA correlated with but was not essential for protection, as demonstrated in studies with IgA-deficient animals.

Conclusion/Significance

Mucosal administration of NP and M2-expressing rAd vectors provided rapid and lasting protection from influenza viruses in a subtype-independent manner. Such vaccines could be used in the interval between emergence of a new virus strain and availability of strain-matched vaccines against it. This strikingly effective single-dose vaccination thus represents a candidate off-the-shelf vaccine for emergency use during an influenza pandemic.     

Sublingual immunization with M2-based vaccine induces broad protective immunity against influenza

Background

The ectodomain of matrix protein 2 (M2e) of influenza A virus is a rationale target antigen candidate for the development of a universal vaccine against influenza as M2e undergoes little sequence variation amongst human influenza A strains. Vaccine-induced M2e-specific antibodies (Abs) have been shown to display significant cross-protective activity in animal models. M2e-based vaccine constructs have been shown to be more protective when administered by the intranasal (i.n.) route than after parenteral injection. However, i.n. administration of vaccines poses rare but serious safety issues associated with retrograde passage of inhaled antigens and adjuvants through the olfactory epithelium. In this study, we examined whether the sublingual (s.l.) route could serve as a safe and effective alternative mucosal delivery route for administering a prototype M2e-based vaccine. The mechanism whereby s.l. immunization with M2e vaccine candidate induces broad protection against infection with different influenza virus subtypes was explored.

Methods and Results

A recombinant M2 protein with three tandem copies of the M2e (3M2eC) was expressed in Escherichia coli. Parenteral immunizations of mice with 3M2eC induced high levels of M2e-specific serum Abs but failed to provide complete protection against lethal challenge with influenza virus. In contrast, s.l. immunization with 3M2eC was superior for inducing protection in mice. In the latter animals, protection was associated with specific Ab responses in the lungs.

Conclusions

The results demonstrate that s.l. immunization with 3M2eC vaccine induced airway mucosal immune responses along with broad cross-protective immunity to influenza. These findings may contribute to the understanding of the M2-based vaccine approach to control epidemic and pandemic influenza infections.     

Cold-adapted influenza and recombinant adenovirus vaccines induce cross-protective immunity against pH1N1 challenge in mice

Background

The rapid spread of the 2009 H1N1 pandemic influenza virus (pH1N1) highlighted problems associated with relying on strain-matched vaccines. A lengthy process of strain identification, manufacture, and testing is required for current strain-matched vaccines and delays vaccine availability. Vaccines inducing immunity to conserved viral proteins could be manufactured and tested in advance and provide cross-protection against novel influenza viruses until strain-matched vaccines became available. Here we test two prototype vaccines for cross-protection against the recent pandemic virus.

Methodology/Principal Findings

BALB/c and C57BL/6 mice were intranasally immunized with a single dose of cold-adapted (ca) influenza viruses from 1977 or recombinant adenoviruses (rAd) expressing 1934 nucleoprotein (NP) and consensus matrix 2 (M2) (NP+M2-rAd). Antibodies against the M2 ectodomain (M2e) were seen in NP+M2-rAd immunized BALB/c but not C57BL/6 mice, and cross-reacted with pH1N1 M2e. The ca-immunized mice did not develop antibodies against M2e. Despite sequence differences between vaccine and challenge virus NP and M2e epitopes, extensive cross-reactivity of lung T cells with pH1N1 peptides was detected following immunization. Both ca and NP+M2-rAd immunization protected BALB/c and C57BL/6 mice against challenge with a mouse-adapted pH1N1 virus.

Conclusion/Significance

Cross-protective vaccines such as NP+M2-rAd and ca virus are effective against pH1N1 challenge within 3 weeks of immunization. Protection was not dependent on recognition of the highly variable external viral proteins and could be achieved with a single vaccine dose. The rAd vaccine was superior to the ca vaccine by certain measures, justifying continued investigation of this experimental vaccine even though ca vaccine is already available. This study highlights the potential for cross-protective vaccines as a public health option early in an influenza pandemic.     

Robust Immunity and Heterologous Protection against Influenza in Mice Elicited by a Novel Recombinant NP-M2e Fusion Protein Expressed in E. coli

Background

The 23-amino acid extracellular domain of matrix 2 protein (M2e) and the internal nucleoprotein (NP) of influenza are highly conserved among viruses and thus are promising candidate antigens for the development of a universal influenza vaccine. Various M2e- or NP-based DNA or viral vector vaccines have been shown to have high immunogenicity; however, high cost, complicated immunization procedures, and vector-specific antibody responses have restricted their applications. Immunization with an NP–M2e fusion protein expressed in Escherichia coli may represent an alternative strategy for the development of a universal influenza vaccine.

Methodology/Principal Findings

cDNA encoding M2e was fused to the 3′ end of NP cDNA from influenza virus A/Beijing/30/95 (H3N2). The fusion protein (NM2e) was expressed in E. coli and isolated with 90% purity. Mice were immunized with recombinant NM2e protein along with aluminum hydroxide gel and/or CpG as adjuvant. NM2e plus aluminum hydroxide gel almost completely protected the mice against a lethal (20 LD₅₀) challenge of heterologous influenza virus A/PR/8/34.

Conclusions/Significance

The NM2e fusion protein expressed in E. coli was highly immunogenic in mice. Immunization with NM2e formulated with aluminum hydroxide gel protected mice against a lethal dose of a heterologous influenza virus. Vaccination with recombinant NM2e fusion protein is a promising strategy for the development of a universal influenza vaccine.     

A Phase I Clinical Study of a Live Attenuated Bordetella pertussis Vaccine - BPZE1; A Single Centre,Double-Blind,Placebo-Controlled,Dose-Escalating Study of BPZE1 Given Intranasally to Healthy Adult Male Volunteers

Background

Acellular pertussis vaccines do not control pertussis. A new approach to offer protection to infants is necessary. BPZE1, a genetically modified Bordetella pertussis strain, was developed as a live attenuated nasal pertussis vaccine by genetically eliminating or detoxifying 3 toxins.

Methods

We performed a double-blind, placebo-controlled, dose-escalating study of BPZE1 given intranasally for the first time to human volunteers, the first trial of a live attenuated bacterial vaccine specifically designed for the respiratory tract. 12 subjects per dose group received 10³, 10⁵ or 10⁷ colony-forming units as droplets with half of the dose in each nostril. 12 controls received the diluent. Local and systemic safety and immune responses were assessed during 6 months, and nasopharyngeal colonization with BPZE1 was determined with repeated cultures during the first 4 weeks after vaccination.

Results

Colonization was seen in one subject in the low dose, one in the medium dose and five in the high dose group. Significant increases in immune responses against pertussis antigens were seen in all colonized subjects. There was one serious adverse event not related to the vaccine. Other adverse events were trivial and occurred with similar frequency in the placebo and vaccine groups.

Conclusions

BPZE1 is safe in healthy adults and able to transiently colonize the nasopharynx. It induces immune responses in all colonized individuals. BPZE1 can thus undergo further clinical development, including dose optimization and trials in younger age groups.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01188512","term_id":"NCT01188512"}}NCT01188512     

Virus-Like Particle Vaccine Protects against 2009 H1N1 Pandemic Influenza Virus in Mice

Background

The 2009 influenza pandemic and shortages in vaccine supplies worldwide underscore the need for new approaches to develop more effective vaccines.

Methodology/Principal Findings

We generated influenza virus-like particles (VLPs) containing proteins derived from the A/California/04/2009 virus, and tested their efficacy as a vaccine in mice. A single intramuscular vaccination with VLPs provided complete protection against lethal challenge with the A/California/04/2009 virus and partial protection against A/PR/8/1934 virus, an antigenically distant human isolate. VLP vaccination induced predominant IgG2a antibody responses, high hemagglutination inhibition (HAI) titers, and recall IgG and IgA antibody responses. HAI titers after VLP vaccination were equivalent to those observed after live virus infection. VLP immune sera also showed HAI responses against diverse geographic pandemic isolates. Notably, a low dose of VLPs could provide protection against lethal infection.

Conclusion/Significance

This study demonstrates that VLP vaccination provides highly effective protection against the 2009 pandemic influenza virus. The results indicate that VLPs can be developed into an effective vaccine, which can be rapidly produced and avoid the need to isolate high growth reassortants for egg-based production.     

Protective efficacy of BCG overexpressing an L,D-transpeptidase against M. tuberculosis infection

Background

M. bovis Bacille Calmette-Guérin (BCG), currently the only available vaccine against tuberculosis (TB), fails to adequately protect individuals from active and latent TB infection. New vaccines are desperately needed to decrease the worldwide burden of TB.

Methods and Findings

We created a recombinant strain of BCG that overproduces an L,D-transpeptidase in order to alter the bacterial peptidoglycan layer and consequently increase the ability of this immunogen to protect against virulent M. tuberculosis (Mtb). We demonstrate that this novel recombinant BCG protects mice against virulent Mtb at least as well as control BCG, as measured by its ability to reduce bacterial burden in lungs and spleen, reduce lung histopathology, and prolong survival. A nutrient starved recombinant BCG preparation, while offering comparable protection, elicited a response characterized by elevated levels of select Th1 cytokines.

Conclusions

Recombinant BCG overexpressing a L,D-transpeptidase that is nutrient starved elicits a stronger Th1 type response and is at least as protective as parent BCG. Results from this study suggest that nutrient starvation treatment of live BCG vaccines should be further investigated as a way to increase host induction of Th-1 related cytokines in the development of experimental anti-TB vaccines.     

Stabilization of Influenza Vaccine Enhances Protection by Microneedle Delivery in the Mouse Skin

Background

Simple and effective vaccine administration is particularly important for annually recommended influenza vaccination. We hypothesized that vaccine delivery to the skin using a patch containing vaccine-coated microneedles could be an attractive approach to improve influenza vaccination compliance and efficacy.

Methodology/Principal Findings

Solid microneedle arrays coated with inactivated influenza vaccine were prepared for simple vaccine delivery to the skin. However, the stability of the influenza vaccine, as measured by hemagglutination activity, was found to be significantly damaged during microneedle coating. The addition of trehalose to the microneedle coating formulation retained hemagglutination activity, indicating stabilization of the coated influenza vaccine. For both intramuscular and microneedle skin immunization, delivery of un-stabilized vaccine yielded weaker protective immune responses including viral neutralizing antibodies, protective efficacies, and recall immune responses to influenza virus. Immunization using un-stabilized vaccine also shifted the pattern of antibody isotypes compared to the stabilized vaccine. Importantly, a single microneedle-based vaccination using stabilized influenza vaccine was found to be superior to intramuscular immunization in controlling virus replication as well as in inducing rapid recall immune responses post challenge.

Conclusions/Significance

The functional integrity of hemagglutinin is associated with inducing improved protective immunity against influenza. Simple microneedle influenza vaccination in the skin produced superior protection compared to conventional intramuscular immunization. This approach is likely to be applicable to other vaccines too.     

Vaccination with M2e-based multiple antigenic peptides: characterization of the B cell response and protection efficacy in inbred and outbred mice

Background

The extracellular domain of the influenza A virus protein matrix protein 2 (M2e) is remarkably conserved between various human isolates and thus is a viable target antigen for a universal influenza vaccine. With the goal of inducing protection in multiple mouse haplotypes, M2e-based multiple antigenic peptides (M2e-MAP) were synthesized to contain promiscuous T helper determinants from the Plasmodium falciparum circumsporozoite protein, the hepatitis B virus antigen and the influenza virus hemagglutinin. Here, we investigated the nature of the M2e-MAP-induced B cell response in terms of the distribution of antibody (Ab) secreting cells (ASCs) and Ab isotypes, and tested the protective efficacy in various mouse strains.

Methodology/Principal Findings

Immunization of BALB/c mice with M2e-MAPs together with potent adjuvants, CpG 1826 oligonucleotides (ODN) and cholera toxin (CT) elicited high M2e-specific serum Ab titers that protected mice against viral challenge. Subcutaneous (s.c.) and intranasal (i.n.) delivery of M2e-MAPs resulted in the induction of IgG in serum and airway secretions, however only i.n. immunization induced anti-M2e IgA ASCs locally in the lungs, correlating with M2-specific IgA in the bronchio-alveolar lavage (BAL). Interestingly, both routes of vaccination resulted in equal protection against viral challenge. Moreover, M2e-MAPs induced cross-reactive and protective responses to diverse M2e peptides and variant influenza viruses. However, in contrast to BALB/c mice, immunization of other inbred and outbred mouse strains did not induce protective Abs. This correlated with a defect in T cell but not B cell responsiveness to the M2e-MAPs.

Conclusion/Significance

Anti-M2e Abs induced by M2e-MAPs are highly cross-reactive and can mediate protection to variant viruses. Although synthetic MAPs are promising designs for vaccines, future constructs will need to be optimized for use in the genetically heterogeneous human population.     

Pre-clinical efficacy and safety of experimental vaccines based on non-replicating vaccinia vectors against yellow fever

Background

Currently existing yellow fever (YF) vaccines are based on the live attenuated yellow fever virus 17D strain (YFV-17D). Although, a good safety profile was historically attributed to the 17D vaccine, serious adverse events have been reported, making the development of a safer, more modern vaccine desirable.

Methodology/Principal Findings

A gene encoding the precursor of the membrane and envelope (prME) protein of the YFV-17D strain was inserted into the non-replicating modified vaccinia virus Ankara and into the D4R-defective vaccinia virus. Candidate vaccines based on the recombinant vaccinia viruses were assessed for immunogenicity and protection in a mouse model and compared to the commercial YFV-17D vaccine. The recombinant live vaccines induced γ-interferon-secreting CD4- and functionally active CD8-T cells, and conferred full protection against lethal challenge already after a single low immunization dose of 10⁵ TCID₅₀. Surprisingly, pre-existing immunity against wild-type vaccinia virus did not negatively influence protection. Unlike the classical 17D vaccine, the vaccinia virus-based vaccines did not cause mortality following intracerebral administration in mice, demonstrating better safety profiles.

Conclusions/Significance

The non-replicating recombinant YF candidate live vaccines induced a broad immune response after single dose administration, were effective even in the presence of a pre-existing immunity against vaccinia virus and demonstrated an excellent safety profile in mice.     

Immunopotentiation of trivalent influenza vaccine when given with VAX102, a recombinant influenza M2e vaccine fused to the TLR5 ligand flagellin

Background

Currently controversy exists about the immunogenicity of seasonal trivalent influenza vaccine in certain populations, especially the elderly. STF2.4×M2e (VAX102) is a recombinant fusion protein that links four copies of the ectodomain of influenza virus matrix protein 2 (M2e) antigen to Salmonella typhimurium flagellin, a TLR5 ligand. The objectives of this study were to assess the feasibility of giving VAX102 and TIV in combination in an effort to achieve greater immunogenicity and to provide cross-protection.

Methodology/Principal Findings

Eighty healthy subjects, 18-49 years old, were enrolled in May and June 2009 in a double-blind, randomized, controlled trial at two clinical sites. Subjects were randomized to receive either TIV + VAX102 or TIV + placebo. Both arms tolerated the vaccines. Pain at the injection site was more severe with TIV + VAX102. Two weeks after immunization the HAI responses to the H1 and H3 antigens of TIV were higher in those that received TIV + VAX102 than in TIV + placebo (309 vs 200 and 269 vs 185, respectively), although statistically non-significant. There was no difference in the HAI of the B antigen. In the TIV + VAX102 arm, the geometric mean M2e antibody concentration was 0.5 µg/ml and 73% seroconverted.

Conclusions/Significance

The combination of TIV + VAX102 has the potential to increase the immune response to the influenza A components of TIV and to provide M2e immunity which may protect against influenza A strains not contained in seasonal TIV.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00921973","term_id":"NCT00921973"}}NCT00921973     

Influenza Vaccine Effectiveness in Preventing Influenza A(H3N2)-Related Hospitalizations in Adults Targeted for Vaccination by Type of Vaccine: A Hospital-Based Test-Negative Study, 2011–2012 A(H3N2) Predominant Influenza Season,Valencia, Spain

Background

Most evidence of the effectiveness of influenza vaccines comes from studies conducted in primary care, but less is known about their effectiveness in preventing serious complications. Here, we examined the influenza vaccine effectiveness (IVE) against hospitalization with PCR-confirmed influenza in the predominant A(H3N2) 2011–2012 influenza season.

Methods

A hospital-based, test-negative study was conducted in nine hospitals in Valencia, Spain. All emergency admissions with a predefined subset of symptoms were eligible. We enrolled consenting adults age 18 and over, targeted for influenza vaccination because of comorbidity, with symptoms of influenza-like-illness within seven days of admission. We estimated IVE as (1-adjusted vaccination odds ratio)*100 after accounting for major confounders, calendar time and recruitment hospital.

Results

The subjects included 544 positive for influenza A(H3N2) and 1,370 negative for influenza admissions. Age was an IVE modifying factor. Regardless of vaccine administration, IVE was 72% (38 to 88%) in subjects aged under 65 and 21% (−5% to 40%) in subjects aged 65 and over. By type of vaccine, the IVE of classical intramuscular split-influenza vaccine, used in subjects 18 to 64, was 68% (12% to 88%). The IVE for intradermal and virosomal influenza vaccines, used in subjects aged 65 and over, was 39% (11% to 58%) and 16% (−39% to 49%), respectively.

Conclusions

The split-influenza vaccine was effective in preventing influenza-associated hospitalizations in adults aged under 65. The intradermal vaccine was moderately effective in those aged 65 and over.     

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.     

Evaluation of In Vitro Cross-Reactivity to Avian H5N1 and Pandemic H1N1 2009 Influenza Following Prime Boost Regimens of Seasonal Influenza Vaccination in Healthy Human Subjects: A Randomised Trial

Introduction

Recent studies have demonstrated that inactivated seasonal influenza vaccines (IIV) may elicit production of heterosubtypic antibodies, which can neutralize avian H5N1 virus in a small proportion of subjects. We hypothesized that prime boost regimens of live and inactivated trivalent seasonal influenza vaccines (LAIV and IIV) would enhance production of heterosubtypic immunity and provide evidence of cross-protection against other influenza viruses.

Methods

In an open-label study, 26 adult volunteers were randomized to receive one of four vaccine regimens containing two doses of 2009-10 seasonal influenza vaccines administered 8 (±1) weeks apart: 2 doses of LAIV; 2 doses of IIV; LAIV then IIV; IIV then LAIV. Humoral immunity assays for avian H5N1, 2009 pandemic H1N1 (pH1N1), and seasonal vaccine strains were performed on blood collected pre-vaccine and 2 and 4 weeks later. The percentage of cytokine-producing T-cells was compared with baseline 14 days after each dose.

Results

Subjects receiving IIV had prompt serological responses to vaccine strains. Two subjects receiving heterologous prime boost regimens had enhanced haemagglutination inhibition (HI) and neutralization (NT) titres against pH1N1, and one subject against avian H5N1; all three had pre-existing cross-reactive antibodies detected at baseline. Significantly elevated titres to H5N1 and pH1N1 by neuraminidase inhibition (NI) assay were observed following LAIV-IIV administration. Both vaccines elicited cross-reactive CD4+ T-cell responses to nucleoprotein of avian H5N1 and pH1N1. All regimens were safe and well tolerated.

Conclusion

Neither homologous nor heterologous prime boost immunization enhanced serum HI and NT titres to 2009 pH1N1 or avian H5N1 compared to single dose vaccine. However heterologous prime-boost vaccination did lead to in vitro evidence of cross-reactivity by NI; the significance of this finding is unclear. These data support the strategy of administering single dose trivalent seasonal influenza vaccine at the outset of an influenza pandemic while a specific vaccine is being developed.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT01044095","term_id":"NCT01044095"}}NCT01044095     

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.     

The Possible Impact of Vaccination for Seasonal Influenza on Emergence of Pandemic Influenza via Reassortment

Background

One pathway through which pandemic influenza strains might emerge is reassortment from coinfection of different influenza A viruses. Seasonal influenza vaccines are designed to target the circulating strains, which intuitively decreases the prevalence of coinfection and the chance of pandemic emergence due to reassortment. However, individual-based analyses on 2009 pandemic influenza show that the previous seasonal vaccination may increase the risk of pandemic A(H1N1) pdm09 infection. In view of pandemic influenza preparedness, it is essential to understand the overall effect of seasonal vaccination on pandemic emergence via reassortment.

Methods and Findings

In a previous study we applied a population dynamics approach to investigate the effect of infection-induced cross-immunity on reducing such a pandemic risk. Here the model was extended by incorporating vaccination for seasonal influenza to assess its potential role on the pandemic emergence via reassortment and its effect in protecting humans if a pandemic does emerge. The vaccination is assumed to protect against the target strains but only partially against other strains. We find that a universal seasonal vaccine that provides full-spectrum cross-immunity substantially reduces the opportunity of pandemic emergence. However, our results show that such effectiveness depends on the strength of infection-induced cross-immunity against any novel reassortant strain. If it is weak, the vaccine that induces cross-immunity strongly against non-target resident strains but weakly against novel reassortant strains, can further depress the pandemic emergence; if it is very strong, the same kind of vaccine increases the probability of pandemic emergence.

Conclusions

Two types of vaccines are available: inactivated and live attenuated, only live attenuated vaccines can induce heterosubtypic immunity. Current vaccines are effective in controlling circulating strains; they cannot always help restrain pandemic emergence because of the uncertainty of the oncoming reassortant strains, however. This urges the development of universal vaccines for prevention of pandemic influenza.     

Vectors based on modified vaccinia Ankara expressing influenza H5N1 hemagglutinin induce substantial cross-clade protective immunity

Background

New highly pathogenic H5N1 influenza viruses are continuing to evolve with a potential threat for an influenza pandemic. So far, the H5N1 influenza viruses have not widely circulated in humans and therefore constitute a high risk for the non immune population. The aim of this study was to evaluate the cross-protective potential of the hemagglutinins of five H5N1 strains of divergent clades using a live attenuated modified vaccinia Ankara (MVA) vector vaccine.

Methodology/Principal Findings

The replication-deficient MVA virus was used to express influenza hemagglutinin (HA) proteins. Specifically, recombinant MVA viruses expressing the HA genes of the clade 1 virus A/Vietnam/1203/2004 (VN/1203), the clade 2.1.3 virus A/Indonesia/5/2005 (IN5/05), the clade 2.2 viruses A/turkey/Turkey/1/2005 (TT01/05) and A/chicken/Egypt/3/2006 (CE/06), and the clade 2.3.4 virus A/Anhui/1/2005 (AH1/05) were constructed. These experimental live vaccines were assessed in a lethal mouse model. Mice vaccinated with the VN/1203 hemagglutinin-expressing MVA induced excellent protection against all the above mentioned clades. Also mice vaccinated with the IN5/05 HA expressing MVA induced substantial protection against homologous and heterologous AH1/05 challenge. After vaccination with the CE/06 HA expressing MVA, mice were fully protected against clade 2.2 challenge and partially protected against challenge of other clades. Mice vaccinated with AH1/05 HA expressing MVA vectors were only partially protected against homologous and heterologous challenge. The live vaccines induced substantial amounts of neutralizing antibodies, mainly directed against the homologous challenge virus, and high levels of HA-specific IFN-γ secreting CD4 and CD8 T-cells against epitopes conserved among the H5 clades and subclades.

Conclusions/Significance

The highest level of cross-protection was induced by the HA derived from the VN/1203 strain, suggesting that pandemic H5 vaccines utilizing MVA vector technology, should be based on the VN/1203 hemagglutinin. Furthermore, the recombinant MVA-HA-VN, as characterized in the present study, would be a promising candidate for such a vaccine.