Influenza Virus H5 DNA Vaccination Is Immunogenic by Intramuscular and Intradermal Routes in Humans

Avian influenza virus causes outbreaks in domestic and wild birds around the world, and sporadic human infections have been reported. A DNA vaccine encoding hemagglutinin (HA) protein from the A/Indonesia/5/05 (H5N1) strain was initially tested in two randomized phase I clinical studies. Vaccine Research Center study 304 (VRC 304) was a double-blinded study with 45 subjects randomized to placebo, 1 mg of vaccine, or 4 mg of vaccine treatment groups (n = 15/group) by intramuscular (i.m.) Biojector injection. VRC 305 was an open-label study to evaluate route, with 44 subjects randomized to intradermal (i.d.) injections of 0.5 mg by needle/syringe or by Biojector or 1 mg delivered as two 0.5-mg Biojector injections in the same deltoid or as 0.5 mg in each deltoid (n = 11/group). Injections were administered at weeks 0, 4, and 8 in both studies. Antibody responses to H5 were assessed by hemagglutination inhibition (HAI) assay, enzyme-linked immunosorbent assay (ELISA), and neutralization assay, and the H5 T cell responses were assessed by enzyme-linked immunospot and intracellular cytokine staining assays. There were no vaccine-related serious adverse events, and the vaccine was well tolerated in all groups. At 1 mg, i.d. vaccination compared to i.m. vaccination induced a greater frequency and magnitude of response by ELISA, but there were no significant differences in the frequency or magnitude of response between the i.d. and i.m. routes in the HAI or neutralization assays. T cell responses were more common in subjects who received the 1- or 4-mg dose i.m. These studies demonstrated that the DNA vaccine encoding H5 is safe and immunogenic and served to define the proper dose and route for further studies. The i.d. injection route did not offer a significant advantage over the i.m. route, and no difference was detected by delivery to one site versus splitting the dose between two sites for i.d. vaccine administration. The 4-mg dose (i.m) was further investigated in prime-boost regimens.     

An H7N1 Influenza Virus Vaccine Induces Broadly Reactive Antibody Responses against H7N9 in Humans

Emerging H7N9 influenza virus infections in Asia have once more spurred the development of effective prepandemic H7 vaccines. However, many vaccines based on avian influenza viruses—including H7—are poorly immunogenic, as measured by traditional correlates of protection. Here we reevaluated sera from an H7N1 human vaccine trial performed in 2006. We examined cross-reactive antibody responses to divergent H7 strains, including H7N9, dissected the antibody response into head- and stalk-reactive antibodies, and tested the in vivo potency of these human sera in a passive-transfer H7N9 challenge experiment with mice. Although only a low percentage of vaccinees induced neutralizing antibody responses against the homologous vaccine strain and also H7N9, we detected strong cross-reactivity to divergent H7 hemagglutinins (HAs) in a large proportion of the cohort with a quantitative enzyme-linked immunosorbent assay. Furthermore, H7N1 vaccination induced antibodies to both the head and stalk domains of the HA, which is in sharp contrast to seasonal inactivated vaccines. Finally, we were able to show that both neutralizing and nonneutralizing antibodies improved in vivo virus clearance in a passive-transfer H7N9 challenge mouse model.     

High Non‐Specific T Lymphocyte Response to the Adjuvanted H1N1 Vaccine in Comparison with the H1N1/H3N2/B‐Brisbane Vaccine without Adjuvant

Shortly after the report of pandemic 2009 influenza A (H1N1), vaccine manufacturers, in conjunction with public agencies, started developing a H1N1 vaccine. In 2009, various approaches were implemented around the globe. The United States and Australia finally approved only non‐adjuvanted H1N1 influenza vaccines, whereas Canada and the EU also approved adjuvanted vaccines. In 2010, seasonal influenza vaccine without adjuvant was again widely accepted in both hemispheres. The addition of adjuvant to the vaccine enhances the immunogenity of the vaccine in the presence of a relatively low amount of antigen. However, it might also induce undesirable non‐specific immune response. For this reason, we conducted a prospective observational study to monitor T cell absolute count and H1N1‐specific immunogenicity after 2009 and 2010 immunization. Fourteen healthy volunteers received the monovalent H1N1 AS03 adjuvanted influenza vaccine (3.5 μg of H1N1 and squalene‐based adjuvant) in October 2009. The immunization was associated with a significant increase in T lymphocyte absolute count (P < 0.0001), reaching abnormal values in 57% of subjects. During this period, none of the subject showed any manifestation of severe viral infection or inflammation. Acute infection by CMV or EBV viruses was also excluded. In October 2010, the same subjects received a seasonal non‐adjuvanted influenza vaccine (15 μg of each: H1N1, H3N2, and B‐Brisbane). However, after 2010 immunization, no change in T lymphocyte absolute count was observed. H1N1‐induced immunogenicity was good for both vaccines. Our results suggest a pronounced non‐specific T cell response after AS03‐adjuvanted 2009 H1N1 vaccination.     

Induction of Local Secretory IgA and Multifunctional CD4+ T‐helper Cells Following Intranasal Immunization with a H5N1 Whole Inactivated Influenza Virus Vaccine in BALB/c Mice

Avian influenza subunit vaccines have been shown to be poorly immunogenic, leading to the re‐evaluation of the immunogenic and dose‐sparing potential of whole virus vaccines. In this study, we investigated the immune responses after one or two doses of intramuscular or intranasal whole inactivated influenza H5N1 virus vaccine in BALB/c mice. Serum samples and nasal washings were collected weekly post‐vaccination and analysed using enzyme‐linked immunosorbent assay (ELISA). Sera were also analysed by the haemagglutination inhibition (HI) assay. Antibody‐secreting cells were measured in lymphocytes from spleen and bone marrow via enzyme‐linked immunospot (ELISPOT). Splenocytes were stimulated in vitro, and T‐helper profiles were measured through multiplex bead assay in the supernatants, or intracellularly by multiparametric flow cytometry. Both vaccine routes induced high HI titres following the second immunization (intramuscular = 370, intranasal = 230). Moreover, the intramuscular group showed significantly higher levels of serum IgG (P < 0.01), IgG1 (P < 0.01) and IgG2a (P < 0.01) following the second vaccine dose, while the intranasal group exhibited significantly higher levels of serum IgA (P < 0.05) and local IgA (P < 0.01) in the nasal washings. Also, IgA antibody‐secreting cells were found in significantly higher numbers in the intranasal group in both the spleen (P < 0.01) and the bone marrow (P < 0.01). Moreover, Th1 (TNF‐α, IL‐2, IFN‐γ) and Th2 (IL‐4, IL‐5, IL‐10) cytokines were expressed by both groups, yet only the intranasal group expressed the Th17 marker IL‐17. As the intranasal vaccines induce local IgA and are easily administered, we suggest the intranasally administered whole virus vaccine as a promising candidate for a pandemic H5N1 vaccine.     

Assessment of Route of Administration and Dose Escalation for an Adenovirus-Based Influenza A Virus (H5N1) Vaccine in Chickens

Highly pathogenic avian influenza (HPAI) virus causes one of the most economically devastating poultry diseases. An HPAI vaccine to prevent the disease in commercial and backyard birds must be effective, safe, and inexpensive. Recently, we demonstrated the efficacy of an adenovirus-based H5N1 HPAI vaccine (Ad5.HA) in chickens. To further evaluate the potential of the Ad5.HA vaccine and its cost-effectiveness, studies to determine the minimal effective dose and optimal route of administration in chickens were performed. A dose as low as 10⁷ viral particles (vp) of adenovirus-based H5N1 vaccine per chicken was sufficient to generate a robust humoral immune response, which correlated with the previously reported level of protection. Several routes of administration, including intratracheal, conjunctival, subcutaneous, and in ovo routes, were evaluated for optimal vaccine administration. However, only the subcutaneous route of immunization induced a satisfactory level of influenza virus-specific antibodies. Importantly, these studies established that the vaccine-induced immunity was cross-reactive against an H5N1 strain from a different clade, emphasizing the potential of cross-protection. Our results suggest that the Ad5.HA HPAI vaccine is safe and effective, with the potential of cross-clade protection. The ease of manufacturing and cost-effectiveness make Ad5.HA an excellent avian influenza vaccine candidate with the ability to protect poultry from HPAI virus infection. Considering the limitations of the influenza vaccine technology currently used for poultry applications, any effort aimed at overcoming those limitations is highly significant.Influenza A virus is a segmented, negative-strand RNA virus that belongs to the family Orthomyxoviridae, which is divided into subtypes based on serological reactions of the two surface glycoproteins, hemagglutinin (HA) and neuraminidase (NA). Thus far, 16 different HA subtypes (H1 to H16) and 9 different NA subtypes (N1 to N9) (11, 34) have been identified. Each of the subtypes has been isolated from waterfowl species, the natural hosts of all known influenza A viruses. These birds are the reservoir for the spread of influenza virus worldwide in wild birds and poultry (19, 34). Avian influenza (AI) virus strains are further classified into low and highly pathogenic avian influenza (LPAI and HPAI, respectively) viruses based on their pathogenicity.Continued outbreaks of HPAI viruses of the H5 and H7 subtypes in poultry in Asia, Europe, Africa, and Canada represent a serious risk for animal and public health worldwide. Avian influenza is one of the greatest concerns for public health that has emerged from an animal reservoir in recent times. Since the late 1990s, the number of outbreaks of avian influenza in poultry has dramatically increased. For example, in 2008-2009, 2,770 outbreaks occurred in Vietnam, 1,143 in Thailand, 1,084 in Egypt, and 219 in Turkey; outbreaks have also occurred in many other countries worldwide (http://www.oie.int/eng/en_index.htm).In 2008-2009, 20 million poultry died or were depopulated because of HPAI outbreaks. This had devastating consequences for the international poultry industry and raised concerns about the potential for transmission to humans and the possible pandemic spread of lethal disease. Culling represents the first line of defense against avian influenza virus; however, continuing outbreaks over the last 6 years revealed that implementation of culling at the farm level was insufficient to halt the spread of disease.Vaccines, in conjunction with other measures of prevention and management, may represent an alternative to preemptive culling to achieve a reduction in the rate of transmission by reducing the susceptibility of healthy flocks at risk. Although vaccination has been recommended by the World Organization for Animal Health (OIE) and the Food and Agriculture Organization (FAO) to control AI, few effective AI vaccines are available (http://www.oie.int/eng/avian_influenza/OIE_FAO_Recom_05.pdf). Conventional inactivated vaccines containing the same viral subtype as field virus (with differing degrees of antigenic similarity) (4, 5, 22), inactivated vaccines generated through reverse genetic techniques (18, 33), and recombinant vaccines (3, 15, 21, 23) have been tested. However, production constraints associated with conventional inactivated influenza virus vaccines that are manufactured in eggs could severely hinder control of an emerging AI virus with pandemic potential (7).We investigated recombinant replication-defective adenoviruses as possible influenza vaccine vehicles for poultry. Recombinant adenovirus-based vaccines are highly effective inducers of both humoral immunity and cellular immunity in mammals and have shown promise as vaccine vehicle candidates against numerous infectious pathogens (2, 12, 14, 24, 32). Previously, we generated Ad5.HA, an E1/E3-deleted human adenovirus serotype 5 (Ad5)-based vector that expresses the codon-optimized hemagglutinin (HA) gene from the influenza A/Vietnam/1203/04 (H5N1) virus (13). The Ad5.HA vaccine induced humoral and cellular immune responses against HA and protected against influenza virus challenges in both mice and chickens. We have now extended our studies to determine the efficacy of Ad5.HA immunization in chickens when administered at different dosages via different routes.     

Long-Term Immunogenicity of an Inactivated Split-Virion 2009 Pandemic Influenza A H1N1 Virus Vaccine with or without Aluminum Adjuvant in Mice

In 2009, a global epidemic of influenza A(H1N1) virus caused the death of tens of thousands of people. Vaccination is the most effective means of controlling an epidemic of influenza and reducing the mortality rate. In this study, the long-term immunogenicity of influenza A/California/7/2009 (H1N1) split vaccine was observed as long as 15 months (450 days) after immunization in a mouse model. Female BALB/c mice were immunized intraperitoneally with different doses of aluminum-adjuvanted vaccine. The mice were challenged with a lethal dose (10× 50% lethal dose [LD₅₀]) of homologous virus 450 days after immunization. The results showed that the supplemented aluminum adjuvant not only effectively enhanced the protective effect of the vaccine but also reduced the immunizing dose of the vaccine. In addition, the aluminum adjuvant enhanced the IgG antibody level of mice immunized with the H1N1 split vaccine. The IgG level was correlated to the survival rate of the mice. Aluminum-adjuvanted inactivated split-virion 2009 pandemic influenza A H1N1 vaccine has good immunogenicity and provided long-term protection against lethal influenza virus challenge in mice.     

Persistence of Antibody to Influenza A/H5N1 Vaccine Virus: Impact of AS03 Adjuvant

The adjuvant AS03 is stockpiled for future formulations with new and existing vaccines for the control of pandemic influenza virus. We previously reported the immunogenicity of an A/H5N1 vaccine extemporaneously mixed with the AS03 adjuvant for 42 days following vaccination. This report extends those findings to 1 year after vaccination.     

High-Level Immunogenicity Is Achieved Vaccine With Adjuvanted Pandemic H1N12009 and Improved With Booster Dosing in a Randomized Trial of HIV-Infected Adults

Abstract

Background: More severe influenza disease and poor vaccine immunogenicity in HIV-infected patients necessitate improved immunization strategies to maximize vaccine efficacy. Methods: A phase III, randomized trial was conducted at 4 Cana-dian sites. Two dosing strategies (standard dose vs standard dose plus booster on day 21) were assessed in HIV patients aged 20 to 59 years during the H1N1₂₀₀₉ pandemic. A single antigen, inactivated split adjuvanted (AS03_A) influenza vaccine (Arepanrix) was utilized. Serum hemagglutination inhibition (HAI) titres were assessed at days 21 and 42 and at month 6. Results: 150 participants received at least one injection. Baseline parameters were similar between groups: 83% male, 85% on HAART, median CD4 = 519 cells/mm³, 84% with HIV RNA < 50 copies/mL. At day 21, seroprotection (HAI ≥1:40) was achieved in 80% (95% CI, 70-89) of participants. Seroconversion occurred in 74% (63–85). Seroprotection and seroconversion were further improved in those randomized to booster dosing: day 42, 94% (85–98) versus 73% (60-83) (P < .01) and 86% (75–93) versus 66% (5–77) (P = .01). Seroprotec-tion was retained in 40% (28–54) of recipients at month 6 with trends toward greater retention of immunity in booster recipients. Conclusion: High-level immunogenicity was achieved with a single dose of this adjuvanted vaccine. Immunogenicity was further improved with booster dosing. Use of this adjuvanted vaccine and booster represent an important approach to increasing immunogenicity in this vaccine hypo-responsive population.     

Influenza A/H5N1 virus infection in humans in Cambodia.

BACKGROUND: Between January 2005 and April 2006, six patients of influenza A/H5N1 virus infection were reported in Cambodia, all with fatal outcome. OBJECTIVES: We describe the virological findings of these six H5N1 patients in association with clinical and epidemiologic findings. STUDY DESIGN: Broncho-alveolar lavage, nasopharyngeal, throat and rectal swabs and sera were cultured for virus isolation and viral load quantified in clinical specimens by real-time RT-PCR. We compared sequences obtained from different body sites within the same patient to detect viral quasi-species. RESULTS: H5N1 virus strains isolated in Cambodia belong to genotype Z, clade 1 viruses. H5N1 viruses were isolated from serum and rectal swab specimens in two patients. The haemagglutinin gene sequences of the virus in different body sites did not differ. Amino acid substitutions known to be associated with a change in virus binding were not observed. CONCLUSION: The high frequency of virus isolation from serum and faecal swabs highlights that H5N1 is likely to be a disseminated infection in humans and this has implications for antiviral treatment, biosafety in clinical laboratories and on risks for nosocomial and human-to-human transmission. There were no tissue-specific adaptive mutations in the HA gene from viruses isolated from different organs.     

Persistence of Immunogenicity of a Monovalent Influenza Virus A/H1N1 2009 Vaccine in Healthy Volunteers

After WHO declared H1N1 pandemic, global vaccination was carried out immediately after much research. However, the data on long-term immunogenicity were lacking. We aimed to investigate the long-term immunogenicity of different H1N1 vaccine dosage groups 24 weeks after vaccination by a randomized clinical trial. A total of 218 participants were stratified into adult (≤60 years old) and elderly (>60 years old) groups. The adults were randomized in a 1:1:1 ratio. The first group received a single dose of vaccine with 15 μg hemagglutination antigen (HA). The other two groups received two doses with 15 μg or 30 μg HA triweekly. The elderly were randomized 1:1 for two doses of 15 or 30 μg HA. We evaluated serologic responses at prevaccination and weeks 3, 6, and 24. We also examined possible associated factors of immunogenicity by multivariate logistic regression analyses. At week 24, seroprotection (anti-HA antibody level ≥ 1:40) remained at 76.8% and 46.2% in the adult and elderly groups, respectively. The adult group had a higher seroprotection rate (odds ratio of 2.98, 95% confidence interval [CI]: 1.21 to 7.36) than the elderly group. There was no statistical difference in seroprotection and seroconversion rates between different adult and elderly dosage groups. Lower immunogenicity in the elderly than in the adults 24 weeks after the vaccination was observed. However, there was no statistically significant difference among different dose groups. Therefore, we suggest only a single vaccination dose of 15 μg HA for adults and two doses of 15 μg HA for the elderly in the future.     

Structural and Functional Changes in Pulmonary Macrophages and Lungs of Mice Infected with Influenza Virus A/H5N1 A/goose/Krasnoozerskoye/627/05

C57Bl/6 mice were intranasally infected with influenza virus A/H5N1 A/goose/Krasnoozerskoye/627/05. The mortality rate of animals reached 70% on day 14 of the disease. The lungs of animals were characterized by necroses, destruction of vessels, hemorrhagic and thrombotic complications, edematous syndrome, and early fibrosis of the interstitium. On days 6-10 after infection, fibrosis was found in the zones of postnecrotic inflammatory infiltration. The expression of lysozyme and myeloperoxidase by pulmonary macrophages was initially increased, but decreased on day 10 of the study. The number of cathepsin D-expressing macrophages was elevated up to the 10th day of examination.     

Safety and Immunogenicity of a Vero Cell Culture-Derived Whole-Virus H5N1 Influenza Vaccine in Chronically Ill and Immunocompromised Patients

The development of vaccines against H5N1 influenza A viruses is a cornerstone of pandemic preparedness. Clinical trials of H5N1 vaccines have been undertaken in healthy subjects, but studies in risk groups have been lacking. In this study, the immunogenicity and safety of a nonadjuvanted cell culture-derived whole-virus H5N1 vaccine were assessed in chronically ill and immunocompromised adults. Subjects received two priming immunizations with a clade 1 A/Vietnam H5N1 influenza vaccine, and a subset also received a booster immunization with a clade 2.1 A/Indonesia H5N1 vaccine 12 to 24 months later. The antibody responses in the two populations were assessed by virus neutralization and single radial hemolysis assays. The T-cell responses in a subset of immunocompromised patients were assessed by enzyme-linked immunosorbent spot assay (ELISPOT). The priming and the booster vaccinations were safe and well tolerated in the two risk populations, and adverse reactions were predominantly mild and transient. The priming immunizations induced neutralizing antibody titers of ≥1:20 against the A/Vietnam strain in 64.2% of the chronically ill and 41.5% of the immunocompromised subjects. After the booster vaccination, neutralizing antibody titers of ≥1:20 against the A/Vietnam and A/Indonesia strains were achieved in 77.5% and 70.8%, respectively, of chronically ill subjects and in 71.6% and 67.5%, respectively, of immunocompromised subjects. The T-cell responses against the two H5N1 strains increased significantly over the baseline values. Substantial heterosubtypic T-cell responses were elicited against the 2009 pandemic H1N1 virus and seasonal A(H1N1), A(H3N2), and B subtypes. There was a significant correlation between T-cell responses and neutralizing antibody titers. These data indicate that nonadjuvanted whole-virus cell culture-derived H5N1 influenza vaccines are suitable for immunizing chronically ill and immunocompromised populations. (This study is registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT00711295","term_id":"NCT00711295"}}NCT00711295.)     

Changes in the Structure of Mouse Kidney in the Acute Period after Infection with Influenza Viruses A/H5N1 and A/H1N1

Bulletin of Experimental Biology and Medicine - Changes in the kidney structure in outbred and inbred male BALB/c mice were analyzed in the acute period after infection with influenza viruses...     

Structural Changes in the Brain of Mice Infected with Influenza A/H5N1 Virus

Structural changes in the brain of outbred mice were studied after infection with infuenza A/H5N1 strain isolated in the Novosibirsk region. High mortality was observed after intranasal infection. Examination of brain specimens revealed vasculopathies with thrombosis of the microcirculatory vessels, pericellular and perivascular edema with multifocal ischemic necrosis, hyperplasia of glial cells, caspase-dependent apoptosis of neurons caused by the cytopathic effect of the virus, and hypercytokinemia.     

Assessment of Antigen-Specific and Cross-Reactive Antibody Responses to an MF59-Adjuvanted A/H5N1 Prepandemic Influenza Vaccine in Adult and Elderly Subjects

Preparedness against an A/H5N1 influenza pandemic requires well-tolerated, effective vaccines which provide both vaccine strain-specific and heterologous, cross-clade protection. This study was conducted to assess the immunogenicity and safety profile of an MF59-adjuvanted, prepandemic influenza vaccine containing A/turkey/Turkey/01/2005 (H5N1) strain viral antigen. A total of 343 participants, 194 adults (18 to 60 years) and 149 elderly individuals (≥61 years), received two doses of the investigational vaccine given 3 weeks apart. Homologous and heterologous antibody responses were analyzed by hemagglutination inhibition (HI), single radial hemolysis (SRH), and microneutralization (MN) assays 3 weeks after administration of the first vaccine dose and 3 weeks and 6 months after the second dose. Immunogenicity was assessed according to European licensure criteria for pandemic influenza vaccines. After two vaccine doses, all three European licensure criteria were met for adult and elderly subjects against the homologous vaccine strain, A/turkey/Turkey/1/2005, when analyzed by HI and SRH assays. Cross-reactive antibody responses were observed by HI and SRH analyses against the heterologous H5N1 strains, A/Indonesia/5/2005 and A/Vietnam/1194/2004, in adult and elderly subjects. Solicited local and systemic reactions were mostly mild to moderate in severity and occurred less frequently in the elderly than in adult vaccinees. In both adult and elderly subjects, MF59-adjuvanted vaccine containing 7.5 μg of A/Turkey strain influenza virus antigen was highly immunogenic, well tolerated, and able to elicit cross-clade, heterologous antibody responses against A/Indonesia and A/Vietnam strains 6 weeks after the first vaccination.