Regulation of antinucleoprotein IgG by systemic vaccination and its effect on influenza virus clearance

Seasonal influenza epidemics recur due to antigenic drift of envelope glycoprotein antigens and immune evasion of circulating viruses. Additionally, antigenic shift can lead to influenza pandemics. Thus, a universal vaccine that protects against multiple influenza virus strains could alleviate the continuing impact of this virus on human health. In mice, accelerated clearance of a new viral strain (cross-protection) can be elicited by prior infection (heterosubtypic immunity) or by immunization with the highly conserved internal nucleoprotein (NP). Both heterosubtypic immunity and NP-immune protection require antibody production. Here, we show that systemic immunization with NP readily accelerated clearance of a 2009 pandemic H1N1 influenza virus isolate in an antibody-dependent manner. However, human immunization with trivalent inactivated influenza virus vaccine (TIV) only rarely and modestly boosted existing levels of anti-NP IgG. Similar results were observed in mice, although the reaction could be enhanced with adjuvants, by adjusting the stoichiometry among NP and other vaccine components, and by increasing the interval between TIV prime and boost. Importantly, mouse heterosubtypic immunity that had waned over several months could be enhanced by injecting purified anti-NP IgG or by boosting with NP protein, correlating with a long-lived increase in anti-NP antibody titers. Thus, current immunization strategies poorly induce NP-immune antibody that is nonetheless capable of contributing to long-lived cross-protection. The high conservation of NP antigen and the known longevity of antibody responses suggest that the antiviral activity of anti-NP IgG may provide a critically needed component of a universal influenza vaccine.     

Protection against divergent influenza H1N1 virus by a centralized influenza hemagglutinin

Influenza poses a persistent worldwide threat to the human population. As evidenced by the 2009 H1N1 pandemic, current vaccine technologies are unable to respond rapidly to this constantly diverging pathogen. We tested the utility of adenovirus (Ad) vaccines expressing centralized consensus influenza antigens. Ad vaccines were produced within 2 months and protected against influenza in mice within 3 days of vaccination. Ad vaccines were able to protect at doses as low as 10(7) virus particles/kg indicating that approximately 1,000 human doses could be rapidly generated from standard Ad preparations. To generate broadly cross-reactive immune responses, centralized consensus antigens were constructed against H1 influenza and against H1 through H5 influenza. Twenty full-length H1 HA sequences representing the main branches of the H1 HA phylogenetic tree were used to create a synthetic centralized gene, HA1-con. HA1-con minimizes the degree of sequence dissimilarity between the vaccine and existing circulating viruses. The centralized H1 gene, HA1-con, induced stronger immune responses and better protection against mismatched virus challenges as compared to two wildtype H1 genes. HA1-con protected against three genetically diverse lethal influenza challenges. When mice were challenged with 1934 influenza A/PR/8/34, HA1-con protected 100% of mice while vaccine generated from 2009 A/TX/05/09 only protected 40%. Vaccination with 1934 A/PR/8/34 and 2009 A/TX/05/09 protected 60% and 20% against 1947 influenza A/FM/1/47, respectively, whereas 80% of mice vaccinated with HA1-con were protected. Notably, 80% of mice challenged with 2009 swine flu isolate A/California/4/09 were protected by HA1-con vaccination. These data show that HA1-con in Ad has potential as a rapid and universal vaccine for H1N1 influenza viruses.     

H3N2 influenza infection elicits more cross-reactive and less clonally expanded anti-hemagglutinin antibodies than influenza vaccination

Background

During the recent H1N1 influenza pandemic, excess morbidity and mortality was seen in young but not older adults suggesting that prior infection with influenza strains may have protected older subjects. In contrast, a history of recent seasonal trivalent vaccine in younger adults was not associated with protection.

Methods and Findings

To study hemagglutinin (HA) antibody responses in influenza immunization and infection, we have studied the day 7 plasma cell repertoires of subjects immunized with seasonal trivalent inactivated influenza vaccine (TIV) and compared them to the plasma cell repertoires of subjects experimentally infected (EI) with influenza H3N2 A/Wisconsin/67/2005. The majority of circulating plasma cells after TIV produced influenza-specific antibodies, while most plasma cells after EI produced antibodies that did not react with influenza HA. While anti-HA antibodies from TIV subjects were primarily reactive with single or few HA strains, anti-HA antibodies from EI subjects were isolated that reacted with multiple HA strains. Plasma cell-derived anti-HA antibodies from TIV subjects showed more evidence of clonal expansion compared with antibodies from EI subjects. From an H3N2-infected subject, we isolated a 4-member clonal lineage of broadly cross-reactive antibodies that bound to multiple HA subtypes and neutralized both H1N1 and H3N2 viruses. This broad reactivity was not detected in post-infection plasma suggesting this broadly reactive clonal lineage was not immunodominant in this subject.

Conclusion

The presence of broadly reactive subdominant antibody responses in some EI subjects suggests that improved vaccine designs that make broadly reactive antibody responses immunodominant could protect against novel influenza strains.     

Immunogenicity and safety of an inactivated quadrivalent influenza vaccine in healthy adults: a phase II,open‐label,uncontrolled trial in Japan

Two antigenically distinct B strain lineages of influenza virus have co‐circulated since the mid‐1980s; however, inactivated trivalent influenza vaccines contain only one B lineage. The mismatch between the circulating and vaccine lineages has been a worldwide issue. In this study, an inactivated quadrivalent influenza vaccine (QIV) candidate containing two B lineages was manufactured and its immunogenicity and safety evaluated in an open‐label, uncontrolled trial. In this phase II trial, 50 subjects aged 20–64 years received two doses of QIV s.c. 1 to 4 weeks apart. Sera were collected pre‐ and post‐vaccination and safety assessed from the first vaccination to 21 ± 7 days after the second vaccination. After the first vaccination, hemagglutination inhibition titers against each strain increased markedly; the seroconversion rate, geometric mean titer ratio and seroprotection rate being 94.0%, 24.93, and 100.0%, respectively, for the A/H1N1pdm09 strain; 94.0%, 12.47, and 98.0%, respectively, for the A/H3N2 strain; 54.0%, 4.99, and 66.0%, respectively, for B/Yamagata strain, and 72.0%, 6.23 and 80.0%, respectively, for the B/Victoria strain, thus fulfilling the criteria of the European Medical Agency's Committee for Medicinal Products for Human Use. Also, the QIV induced sufficient single radial hemolysis and neutralizing antibodies against all four vaccine strains. No noteworthy adverse events were noted. The results of this trial demonstrate that QIV is well tolerated and immunogenic for each strain, suggesting that QIV potentially improves protection against influenza B by resolving the issue of B lineage mismatch.     

Differing Epidemiological Dynamics of Influenza B Virus Lineages in Guangzhou,Southern China, 2009-2010

The epidemiological and evolutionary dynamics of the two cocirculating lineages of influenza B virus, Victoria and Yamagata, are poorly understood, especially in tropical or subtropical areas of Southeast Asia. We performed a phylogenetic analysis of the hemagglutinin (HA) and neuraminidase (NA) sequences of influenza B viruses isolated in Guangzhou, a southern Chinese city, during 2009 to 2010 and compared the demographic and clinical features of infected patients. We identified multiple viral introductions of Victoria strains from both Chinese and international sources, which formed two phylogenetically and antigenically distinct clades (Victoria 1 and 2), some of which persisted between seasons. We identified one dominant Yamagata introduction from outside China during 2009. Our phylogenetic analysis reveals the occurrence of reassortment events among the Victoria and Yamagata lineages and also within the Victoria lineage. We found no significant difference in clinical severity by influenza B lineage, with the exceptions that (i) the Yamagata lineage infected older people than either Victoria lineage and (ii) fewer upper respiratory tract infections were caused by the Victoria 2 than the Victoria 1 clade. Overall, our study reveals the complex epidemiological dynamics of different influenza B lineages within a single geographic locality and has implications for vaccination policy in southern China.     

Immunogenicity and cross-reactivity of 2009-2010 inactivated seasonal influenza vaccine in US adults and elderly

The campaign of 2009-2010 Northern Hemisphere seasonal vaccination was concurrent with the 2009 H1N1 pandemic. Using a hemagglutination inhibition (HAI) assay, we evaluated the immunogenicity and cross-reactivity of 2009-2010 inactivated trivalent influenza vaccine (TIV) in US adult and elderly populations. Vaccination of TIV resulted in a robust boost on the antibody response of all subjects to seasonal A/Brisbane/59/2007 (H1N1) and A/Uruguay/716/2007 (H3N2) with over 70% of recipients reaching a seroprotective titer of 40. B/Brisbane/60/2008 was the least immunogenic among the three seasonal vaccine strains with <30% of TIV recipients reaching a seroprotective titer of 40. TIV vaccination also induced a moderate boost on the pandemic specific antibody responses. Twenty-four percent of adults and 36% of elderly reached a seroprotective HAI titer of 40 or more against pandemic A/South Carolina/18/2009 (H1N1) after receiving TIV compared to 4% and 7% at the beginning of vaccination, respectively. In addition, 22% of adults and 34% of elderly showed an increase of 4-fold or more in A/South Carolina/18/2009 specific HAI titers after TIV vaccination. The pandemic specific cross-reactive antibodies strongly correlated with the post-vaccination HAI titers against the seasonal H3N2 vaccine strain in all subjects.     

Epidemiological and Evolutionary Dynamics of Influenza B Viruses in Malaysia, 2012-2014

Epidemiological and evolutionary dynamics of influenza B Victoria and Yamagata lineages remained poorly understood in the tropical Southeast Asia region, despite causing seasonal outbreaks worldwide. From 2012–2014, nasopharyngeal swab samples collected from outpatients experiencing acute upper respiratory tract infection symptoms in Kuala Lumpur, Malaysia, were screened for influenza viruses using a multiplex RT-PCR assay. Among 2,010/3,935 (51.1%) patients infected with at least one respiratory virus, 287 (14.3%) and 183 (9.1%) samples were tested positive for influenza A and B viruses, respectively. Influenza-positive cases correlate significantly with meteorological factors—total amount of rainfall, relative humidity, number of rain days, ground temperature and particulate matter (PM10). Phylogenetic reconstruction of haemagglutinin (HA) gene from 168 influenza B viruses grouped them into Yamagata Clade 3 (65, 38.7%), Yamagata Clade 2 (48, 28.6%) and Victoria Clade 1 (55, 32.7%). With neuraminidase (NA) phylogeny, 30 intra-clade (29 within Yamagata Clade 3, 1 within Victoria Clade 1) and 1 inter-clade (Yamagata Clade 2-HA/Yamagata Clade 3-NA) reassortants were identified. Study of virus temporal dynamics revealed a lineage shift from Victoria to Yamagata (2012–2013), and a clade shift from Yamagata Clade 2 to Clade 3 (2013–2014). Yamagata Clade 3 predominating in 2014 consisted of intra-clade reassortants that were closely related to a recent WHO vaccine candidate strain (B/Phuket/3073/2013), with the reassortment event occurred approximately 2 years ago based on Bayesian molecular clock estimation. Malaysian Victoria Clade 1 viruses carried H274Y substitution in the active site of neuraminidase, which confers resistance to oseltamivir. Statistical analyses on clinical and demographic data showed Yamagata-infected patients were older and more likely to experience headache while Victoria-infected patients were more likely to experience nasal congestion and sore throat. This study describes the evolution of influenza B viruses in Malaysia and highlights the importance of continuous surveillance for better vaccination policy in this region.     

Purified influenza virus hemagglutinin and neuraminidase are equivalent in stimulation of antibody response but induce contrasting types of immunity to infection.

BALB/c mice immunized with graded doses of chromatographically purified hemagglutinin (HA) and neuraminidase (NA) antigens derived from A/Hong Kong/1/68 (H3N2) influenza virus demonstrated equivalent responses when HA-specific and NA-specific serum antibodies were measured by enzyme-linked immunosorbent assays (ELISAs). Antibody responses measured by hemagglutination inhibition or neuraminidase inhibition titrations showed similar kinetic patterns, except for more rapid decline in hemagglutination inhibition antibody. Injection of mice with either purified HA or NA resulted in immunity manifested by reduction in pulmonary virus following challenge with virus containing homologous antigens. However, the nature of the immunity induced by the two antigens differed markedly. While HA immunization with all but the lowest doses of antigen prevented manifest infection, immunization with NA was infection-permissive at all antigen doses, although reduction in pulmonary virus was proportional to the amount of antigen administered. The immunizing but infection-permissive effect of NA immunization over a wide range of doses is in accord with results of earlier studies with mice in which single doses of NA and antigenically hybrid viruses were used. The demonstrable immunogenicity of highly purified NA as a single glycoprotein without adjuvant offers a novel infection-permissive approach with potentially low toxicity for human immunization against influenza virus.     

南京市2011年乙型流感血凝素基因分子特征分析

[目的]分析2011年南京市乙型流感病毒的血凝素(HA)分子学特征.[方法]选择7株2011年南京市不同时间段有代表性的乙型流感毒株进行HA基因序列测定,通过生物信息学方法对HA分子学特征进行分析.[结果]7株乙型流感毒株分为两个系,4株为Victoria,3株为Yamagata;与2011年度疫苗株相比,Victoria和Yamagata系毒株分别在抗原位点146、197和116、198发生了氨基酸替换;其中197和198位点分别是Victoria和Yamagata毒株的受体结合位点,由于上述位点的替换使得Victoria系/Yamagata系毒株分别在197/196位增加了一个潜在的糖基化位点.[结论]2011年南京市乙型流感Victoria 系和Yamagata系病毒同时存在,Victoria/Yamagata毒株197/198位点的氨基酸替换,值得做进一步的探讨.     

Influence of prior influenza vaccination on antibody and B-cell responses

Currently two vaccines, trivalent inactivated influenza vaccine (TIV) and live attenuated influenza vaccine (LAIV), are licensed in the USA. Despite previous studies on immune responses induced by these two vaccines, a comparative study of the influence of prior influenza vaccination on serum antibody and B-cell responses to new LAIV or TIV vaccination has not been reported. During the 2005/6 influenza season, we quantified the serum antibody and B-cell responses to LAIV or TIV in adults with differing influenza vaccination histories in the prior year: LAIV, TIV, or neither. Blood samples were collected on days 0, 7-9 and 21-35 after immunization and used for serum HAI assay and B-cell assays. Total and influenza-specific circulating IgG and IgA antibody secreting cells (ASC) in PBMC were detected by direct ELISPOT assay. Memory B cells were also tested by ELISPOT after polyclonal stimulation of PBMC in vitro. Serum antibody, effector, and memory B-cell responses were greater in TIV recipients than LAIV recipients. Prior year TIV recipients had significantly higher baseline HAI titers, but lower HAI response after vaccination with either TIV or LAIV, and lower IgA ASC response after vaccination with TIV than prior year LAIV or no vaccination recipients. Lower levels of baseline HAI titer were associated with a greater fold-increase of HAI titer and ASC number after vaccination, which also differed by type of vaccine. Our findings suggest that the type of vaccine received in the prior year affects the serum antibody and the B-cell responses to subsequent vaccination. In particular, prior year TIV vaccination is associated with sustained higher HAI titer one year later but lower antibody response to new LAIV or TIV vaccination, and a lower effector B-cell response to new TIV but not LAIV vaccination.     

Single immunization with MF59-adjuvanted inactivated whole-virion H7N9 influenza vaccine provides early protection against H7N9 virus challenge in mice

H7N9 influenza infection in humans would result in severe respiratory illness. Vaccination is the best way to prevent influenza virus. In this paper, we investigated the effect of early protection provided by inactivated whole-virion H7N9 influenza vaccine in a mouse model.Mice were immunized intramuscularly once with different doses of inactivated whole-virion H7N9 influenza vaccine alone or in combination with MF59 adjuvant. Specific IgM and IgG antibody titers in sera of mice were detected by ELISA 3, 5 and 7days after immunization. To evaluate the early protection provided by the vaccine, mice were challenged with lethal dose (40LD₅₀) of homologous virus 3, 5 and 7 days after immunization respectively. The survival rate and body weight change of mice during 21 days after challenge and the residual lung virus titer on 3rd day after challenge were determined. The results demonstrated that mice could obtain effective protection 3 days after immunization with the vaccine at a high dose, and 5–7 days after immunization even at a low dose. Thus early immune responses induced by inactivated whole-virion H7N9 vaccine could provide effective protection.     

Seasonal trivalent inactivated influenza vaccine protects against 1918 Spanish influenza virus infection in ferrets

The influenza virus H1N1 pandemic of 1918 was one of the worst medical catastrophes in human history. Recent studies have demonstrated that the hemagglutinin (HA) protein of the 1918 virus and 2009 H1N1 pandemic virus [A(H1N1)pdm09], the latter now a component of the seasonal trivalent inactivated influenza vaccine (TIV), share cross-reactive antigenic determinants. In this study, we demonstrate that immunization with the 2010-2011 seasonal TIV induces neutralizing antibodies that cross-react with the reconstructed 1918 pandemic virus in ferrets. TIV-immunized ferrets subsequently challenged with the 1918 virus displayed significant reductions in fever, weight loss, and virus shedding compared to these parameters in nonimmune control ferrets. Seasonal TIV was also effective in protecting against the lung infection and severe lung pathology associated with 1918 virus infection. Our data demonstrate that prior immunization with contemporary TIV provides cross-protection against the 1918 virus in ferrets. These findings suggest that exposure to A(H1N1)pdm09 through immunization may provide protection against the reconstructed 1918 virus which, as a select agent, is considered to pose both biosafety and biosecurity threats.     

Molecular Epidemiology and Phylogenetic Analyses of Influenza B Virus in Thailand during 2010 to 2014

Influenza B virus remains a major contributor to the seasonal influenza outbreak and its prevalence has increased worldwide. We investigated the epidemiology and analyzed the full genome sequences of influenza B virus strains in Thailand between 2010 and 2014. Samples from the upper respiratory tract were collected from patients diagnosed with influenza like-illness. All samples were screened for influenza A/B viruses by one-step multiplex real-time RT-PCR. The whole genome of 53 influenza B isolates were amplified, sequenced, and analyzed. From 14,418 respiratory samples collected during 2010 to 2014, a total of 3,050 tested positive for influenza virus. Approximately 3.27% (471/14,418) were influenza B virus samples. Fifty three isolates of influenza B virus were randomly chosen for detailed whole genome analysis. Phylogenetic analysis of the HA gene showed clusters in Victoria clades 1A, 1B, 3, 5 and Yamagata clades 2 and 3. Both B/Victoria and B/Yamagata lineages were found to co-circulate during this time. The NA sequences of all isolates belonged to lineage II and consisted of viruses from both HA Victoria and Yamagata lineages, reflecting possible reassortment of the HA and NA genes. No significant changes were seen in the NA protein. The phylogenetic trees generated through the analysis of the PB1 and PB2 genes closely resembled that of the HA gene, while trees generated from the analysis of the PA, NP, and M genes showed similar topology. The NS gene exhibited the pattern of genetic reassortment distinct from those of the PA, NP or M genes. Thus, antigenic drift and genetic reassortment among the influenza B virus strains were observed in the isolates examined. Our findings indicate that the co-circulation of two distinct lineages of influenza B viruses and the limitation of cross-protection of the current vaccine formulation provide support for quadrivalent influenza vaccine in this region.     

A monoclonal antibody targeting a highly conserved epitope in influenza B neuraminidase provides protection against drug resistant strains

All influenza viral neuraminidases (NA) of both type A and B viruses have only one universally conserved sequence located between amino acids 222–230. A monoclonal antibody against this region has been previously reported to provide broad inhibition against all nine subtypes of influenza A NA; yet its inhibitory effect against influenza B viral NA remained unknown. Here, we report that the monoclonal antibody provides a broad inhibition against various strains of influenza B viruses of both Victoria and Yamagata genetic lineage. Moreover, the growth and NA enzymatic activity of two drug resistant influenza B strains (E117D and D197E) are also inhibited by the antibody even though these two mutations are conformationally proximal to the universal epitope. Collectively, these data suggest that this unique, highly-conserved linear sequence in viral NA is exposed sufficiently to allow access by inhibitory antibody during the course of infection; it could represent a potential target for antiviral agents and vaccine-induced immune responses against diverse strains of type B influenza virus.     

Evaluation of the efficacy of split-product trivalent A(H1N1), A(H3N2), and B influenza vaccines: reactogenicity, immunogenicity and persistence of antibodies following two doses of vaccines

The reactogenicity and immunogenicity of Tween-ether split trivalent A(H1N1), A(H3N2), and B influenza vaccine in primary school children aged seven to 12 years, and the persistence of antibodies following two doses of vaccine were studied during 1980-1984. Adverse reactions were infrequent, and, even when reported, were chiefly local ones, mild in nature and of short duration. Most of the reactions were less frequent after the second dose than after the first dose. Most of the systemic reactions occurred during the intervaccination period with almost equal frequency, indicating that careful consideration is required to judge whether they were induced by vaccination or not. This vaccine had induced adequate hemagglutination inhibiting (HAI) antibody because the geometric mean titers (GMTs) of the vaccinees were two- to eightfold higher than those of the nonvaccinees to any of the vaccine antigens following two doses of vaccine. In general, the responses to A(H3N2) virus were the best among the vaccine antigens through the four vaccination seasons, but there was a tendency to show a poorer response to the same type (or subtype) of virus antigen as the causative one during a protracted epidemic. The antibodies induced by either vaccination or natural infection were shown to persist for less than a year, supporting the recommendation for annual vaccination.     

Applicability of a sensitive duplex real-time PCR assay for identifying B/Yamagata and B/Victoria lineages of influenza virus from clinical specimens

Type B influenza virus is one of the major epidemic strains and responsible for considerable mortality and morbidity. Rapidly and accurately identifying different influenza B virus lineages, i.e., B/Yamagata (B/Y) and B/Victoria (B/V), is desirable during the flu season. However, the available rapid techniques lack sensitivity, and the usual methods for identifying influenza viruses require expansion of virus in tissue culture or embryonated hen's eggs. Thus, we developed several sets of primer pairs that were able to detect and distinguish B/Y and B/V in a single real-time PCR assay. Used in conjunction with two sets of specific primers that exhibited purine at 3' end of at least one primer targeting on HA gene of B/Y and B/V lineages allows us to accurately identify approximately 10(2) copies per microliter for B/Y and B/V with intra- and inter-assay coefficient of variation (CV) <4%. When it was used to test 17,765 throat swab specimens obtained in the 2006-2010 influenza surveillance season, this method was comparable to hemagglutination inhibition assay in detection, typing and subtyping of influenza viruses with 100% true-negative (specificity) and 100% true-positive (sensitivity). Taken together, this method provides sensitive and robust tool for routine diagnosis and on-time epidemiological examination for WHO decisions on vaccine composition.     

Efficacy of seasonal pandemic influenza hemagglutinin DNA vaccines delivered by electroporation against aseasonal H1N1 virus challenge in mice

Prophylactic DNA vaccines against the influenza virus are promising alternatives to conventional vaccines. In this study, we generated two candidate gene-based influenza vaccines encoding either the seasonal or pandemic hemagglutinin antigen (HA) from the strains A/New Caledonia/20/99 (H1N1) (pV1A5) and A/California/04/2009 (H1N1) (pVEH1), respectively. After verifying antigen expression, the immunogenicity of the vaccines delivered intramuscularly with electroporation was tested in a mouse model. Sera of immunized animals were tested in hemagglutination inhibition assays and by ELISA for the presence of HA-specific antibodies. HA-specific T-cells were also measured in IFN-γ ELISpot assays. The protective efficacy of the candidate influenza vaccines was evaluated by measuring mortality rates and body weight after a challenge with 100 LD(50) of mouse-adapted A/New Caledonia/20/99 (H1N1). Mice immunized with either one of the two vaccines showed significantly higher T cell and humoral immune responses (P<0.05) than the pVAX1 control group. Additionally, the pV1A5 vaccine effectively protected the mice against a lethal homologous mouse-adapted virus challenge with a survival rate of 100% compared with a 40% survival rate in the pVEH1 vaccinated group (P<0.05). Our study indicates that the seasonal influenza DNA vaccine completely protects against the homologous A/New Caledonia/20/99 virus (H1N1), while the pandemic influenza DNA vaccine only partially protects against this virus.     

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.     

Characterization of cross protection of Swine-Origin Influenza Virus (S-OIV) H1N1 and reassortant H5N1 influenza vaccine in BALB/c mice given a single-dose vaccination

Background

Influenza virus has antigen drift and antigen shift effect, vaccination with some influenza vaccine might not induce sufficient immunity for host to the threat of other influenza virus strains. S-OIV H1N1 and H5N1 influenza vaccines in single-dose immunization were evaluated in mice for cross protection to the challenge of A/California/7/2009 H1N1 or NIBRG-14 H5N1 virus.

Results

Both H1N1 and H5N1 induced significant homologous IgG, HAI, and microneutralization antibody responses in the mice, while only vaccines plus adjuvant produced significant heterogeneous IgG and HAI antibody responses. Both alum and MPLA adjuvants significantly reduced the S-OIV H1N1 vaccine dose required to elicit protective HAI antibody titers from 0.05 μg to 0.001 μg. Vaccines alone did not protect mice from challenge with heterogeneous influenza virus, while H5N1 vaccine plus alum and MPLA adjuvants did. Mouse body weight loss was also less significant in the presence of adjuvant than in the vaccine without adjuvant. Furthermore, both H1N1 and H5N1 lung viral titers of immunized mice were significantly reduced post challenge with homologous viruses.

Conclusion

Only in the presence of MPLA adjuvant could the H5N1 vaccine significantly reduce mouse lung viral titers post H1N1 virus challenge, and not vice versa. MPLA adjuvant induced cross protection with a single dose vaccination to the challenge of heterogeneous influenza virus in mice. Lung viral titer seemed to be a better indicator compared to IgG, neutralization antibody, and HAI titer to predict survival of mice infected with influenza virus.