Immunogenicity and Safety of Pandemic Influenza H5N1 Vaccines in Healthy Adults through Meta-Analysis

Influenza is a significant cause of respiratory infection and one of the primary causes of infection-related deaths among humans. Avian influenza, known informally as avian flu or bird flu, refers to influenza caused by viruses adapted to birds. Recent influenza research into the genes of the Spanish flu virus shows genes from both human and avian strains. This mixture of genes could create a new virus (via reassortment) that could cause an antigenic shift to a new influenza A virus subtype from which most people have little or no immune protection [1,2]. Avian influenza strains are divided into two types based on their pathogenicity: high pathogenicity (HP) or low pathogenicity (LP) [3]. The most well-known highly pathogenic avian influenza (HPAI) strain, H5N1, appeared in China in 1996, and LP strains have been found in North America [4,5]. The highly pathogenic influenza A virus subtype H5N1 is an emerging avian influenza virus that is causing global concern as a potential pandemic threat. H5N1 has killed millions of poultry in a growing number of countries throughout Asia, Europe and Africa. Health experts are concerned that the coexistence of human flu viruses and avian flu viruses (especially H5N1) will provide an opportunity for genetic material to be exchanged between species-specific viruses, possibly creating a new virulent influenza strain that is easily transmittable and lethal to humans. The mortality rate for humans with H5N1 is 60% [6,7,8,9,10].

Since the first human H5N1 outbreak that occurred in 1997, there has been an increasing number of HPAI H5N1 bird-to-human transmissions, leading to clinically severe and fatal human infections. The first known transmission of H5N1 to a human occurred in Hong Kong in 1997, when there was an outbreak of 18 human cases; 6 deaths were confirmed. None of the infected people worked with poultry. After culling all of the poultry in the area, no more cases were diagnosed [11,12]. In 2006, the first human-to-human transmission likely occurred when 7 members of a family in Sumatra became infected after contact with a family member who had worked with infected poultry [13,14,15].

The H5N1 outbreak in Thailand caused massive economic losses, especially among poultry workers. Infected birds were culled and slaughtered. The public lost confidence in poultry products, thus decreasing the consumption of chicken products. The outbreak also elicited a ban from importing countries. There were, however, factors that aggravated the spread of the virus, including bird migration, cool temperatures (which increases virus survival) and several ongoing festivals during that time [16,17,18].

Additionally, several domesticated species have been infected with and shown symptoms of H5N1 viral infection, including cats, dogs, ferrets, pigs, and birds [19,20,21]. Vaccines for poultry have been formulated against several of the avian H5N1 influenza varieties. Control measures for HPAI encourage mass vaccinations of poultry. The World Health Organization (WHO) has compiled a list of known clinical trials of pandemic influenza prototype vaccines, including those against H5N1 [22,23]. In some countries still at a high risk for the spread of HPAI, there is compulsory strategic vaccination, though vaccine supply shortages remain a problem. In the event of a pandemic, the demand for Avian influenza vaccines (AIV) to prevent and control the avian influenza infection is substantial [24]. However, current manufacturing processes for AIV require four to six months [17]. The use of adjuvants might be a very useful method for lower consumption and savings.

An adjuvantis a pharmacological or immunological agent that modifies the effect of other agents. Adjuvants may be added to a vaccine to modify the immune response by boosting the response to produce more antibodies and longer-lasting protection, thus minimizing the amount of injected antigenic material. Adjuvants may also be used to enhance the efficacy of a vaccine by helping to modify the immune response to particular types of immune system cells: for example, by activating T cells instead of antibody-secreting B cells, depending on the purpose of the vaccine. The most common adjuvant for H5N1 AlV is aluminium-adjuvant vaccines. However, according to some publications, aluminium adjuvant cannot reduce the required vaccine dose, even with no ceiling effect [18]. If the AIV dose was below 7.5 µg, there was no protection effect in humans when aluminium adjuvant was used [19,21]. Thus, scientists attempted to use novel oil adjuvants (e.g., MF59 and AS03) that could halve the consumption of AIV [18].

There are few reviews and meta-analyses to assess the immunogenicity and safety of a low AIV dose when administered in conjunction with novel oil adjuvants. This study aimed to analyse the available data on this topic.

We performed a literature search by searching for "H5N1" and "vaccine" using the Cochrane Central Register of Controlled Trials (CENTRAL), Medline, Excerpata Medica Database (EMBASE) and China National Knowledge Infrastructure (CNKI) up through April 2016. No limitation was placed on the language or year of publication. In the online databases, we used the following keywords: influenza vaccine, inactivated vaccine, H5N1 avian influenza, H5N1 HPAI, H5N1 influenza A virus and H5N1 highly pathogenic avian influenza. Simultaneously, we made an index of the different types of adjuvants. We also searched clinicaltrails. gov for required information. We collected not only primary publications but also their reference lists for additional publications. Additionally, we searched databases using the term "AIV" together with the first author of each article and examined the 5 most recent related links to all articles in the PubMed database.

The inclusion criteria were selected as follows: randomized trials research of H5N1 AIV, use of adjuvant (or not), subjects aged from 18 to 60 years old, analysis of the change in antibody levels, and vaccine-related adverse reactions (e.g., fever, redness, shivering, malaise, ecchymosis, local pain and headache).

The immunogenicity of AIV was assessed according to the criteria from the European Medicines Agency (EMA). The EMA stated that AIV could be effective if it demonstrated one of the following: a seroconversion rate > 40% or a seroprotection rate > 70%. The safety assessment of AIV included systemic and local adverse events within 3 days post-vaccination.

Two independent staff extracted data from all included studies, including vaccine-related adverse reactions, assessment of immunogenicity and safety, use of adjuvants, study design and population, influenza antibody detection methods and the first author and year of the reference. Any disagreements or discrepancies were resolved by discussion or by consulting an expert committee at the Institute of Medical Biology.

The database was collected using Microsoft Excel. Stata 12.0 and Revman 5.0 software were used for the statistical analyses. The AIV dose associated with adjuvant use was assessed by calculating the merger ratio (odds ratio, OR), and the 95% confidence area (confidence interval, 95% CI) was evaluated. Publication bias was evaluated using the Revman 5.0 software and is shown in the risk of bias summary diagram (Fig. 1).

After searching on PubMed, the China National Knowledge Infrastructure (CNKI), the EMBASE database and the Cochrane database, we found 357 articles, which included 191 repetitions (Fig. 2). We then built a database of inclusion research. After reading the full text of our sub-group discussion, there were 24 theses [6,7,8,9,10,11,13,14,15,19,20,21,22,23,24,25,26,27,28,29,30,31,32,33] focusing on the dose consumption and safety of novel H5N1 AIV, two articles with an in-depth analysis regarding the cross-reactive immunogenicity of H5N1 AIV [34,35], and two articles on a long-term immunogenicity analysis of a vaccine and the administration procedures of a vaccine [36,37]. Finally, we identified 24 articles consistent with our inclusion criteria and used them for the meta-analysis. We briefly summarized the characteristics of all the included studies in Table 1. For the 24 publications of randomized trails, all the subjects were aged from 18 to 60 years. The technology and methodology used for AIV antibody detection included haemagglutinin inhibition (HI) and the microneutralization (MN) test.

We found a different concentration gradient within the groups that used different AIV doses, which generally used <3 µg, 3.75 µg, 7.5 µg, and 15 µg of AIV. Evaluation of the immunogenicity and safety of AIV was performed in a subsequent analysis using Revman 5.0 and Stata 12.0 software.

In this systematic analysis, we collected the experimental data and divided them into many different subgroups according to different research topics using Stata 12.0. The RR value and 95% CI were calculated. The random-effect method model was used. We found that when the dose of H5N1 AIV was between 3.75 µg and 7.5 µg HA antigen, the studies applied for a trial extension. Characteristics of the included trails are shown in Table 1.

The adjuvant played an important role in producing effective antibodies at sufficiently high levels for the vaccines. The adjuvant could be a significant factor in the use of AIV. However, aluminium adjuvants were used at the same vaccine dose as the no-adjuvant group and induced the same immune effects (Table 2 and Table 3). This result shows that an aluminium adjuvant is not the most appropriate choice for AIV. AIV requires other novel types of adjuvant. This result is consistent with another meta-analysis [18].

Consistent with this finding, novel adjuvants (MF59 and AS03) were used with a smaller dose of vaccine than aluminium and stimulated the body to produce effective antibodies. There was no significant difference between the 1.9 µg and 3.75 µg vaccine dosage when used with the AS03 adjuvant. Thus, the AS03 adjuvant worked with an AIV dose as low as 1.9 µg, which could reduce consumption [11,12,22,23]. With AS03 synergy even the lowest amount of AIV (1.9 µg) produced enough MN antibody titer (RR=0.940, CI% (0.860- 1.028)). However, when combined with the AS03 adjuvant, a dose of 1.9 µg of vaccine produced a smaller HI titer than 3.75 µg of vaccine (Table 3).

The MF59 adjuvant had an obvious vaccine dose-dependent trend. If the vaccine dosage was greater than 3.75 µg, it could achieve a similar effect to the AS03 adjuvant; however, less than 3.75 µg of vaccine did not produce an effect in the subject.

All of the H5N1 AIVs that were used were well tolerated without serious adverse reactions. In the safety assessment of H5N1 AIV, serious vaccine-related adverse events (AEs) were not found. This analysis also included different types of adjuvants. Some reported AEs were mild to moderate and included fever, headache, and injection point pain. However, we could not determine specific reasons for these AEs. It should be noted that there was no evidence to prove the relationship between a higher dose and an increased risk of adverse events in the non-adjuvant group (Table 4). We concluded that the studied dosages of H5N1 AIV were well tolerated.

We compared the inactivated whole virus vaccines with subunit and split virus vaccines. The process of producing whole virus vaccines does not require purification and splitting, which can save time in the AIV production cycle. There was no significant difference between the three forms in the assessment of results for the immune effects. Furthermore, all three forms of the virus particles had the same dose-dependent immunogenicity trend (Table 5). The effects of adjuvant on whole virion vaccine immunity were consistent with the previous analysis.

One strain of HPAI (H5N1) is spreading globally after first appearing in Asia. According to the World Health Organization and the United Nations Food and Agriculture Organization (FAO), H5N1 pathogenicity is gradually continuing to rise in endemic areas, but avian influenza in farmed birds is being held in check by vaccination.

Eleven outbreaks of H5N1 were reported worldwide in June 2008 in five countries (China, Egypt, Indonesia, Pakistan and Vietnam), compared to 65 outbreaks in June 2006 and 55 in June 2007. The global HPAI situation significantly improved in the first half of 2008, but the FAO reports that imperfect disease surveillance systems cause the occurrence of the virus to remain underestimated and under-reported. In July 2013, the WHO announced a total of 630 confirmed human cases that resulted in the deaths of 375 people since 2003 [3,4,5,38]. Several H5N1 vaccines have been developed, approved, and stockpiled by a number of countries, including the United States (in its National Stockpile), Britain, France, Canada, and Australia, for use in an emergency.

There are several H5N1 vaccines for avian H5N1 varieties, but the continual mutation of H5N1 limits their present-day use. Although vaccines can sometimes provide cross-protection against related flu strains, the best protection is provided by a vaccine specifically produced for a future pandemic flu virus strain. However, pre-pandemic vaccines have been created, refined and tested, and do show some promise in furthering both research and preparedness for the next pandemic. Vaccine manufacturing companies are being encouraged to increase capacity so that if a pandemic vaccine is needed, facilities will be available for the rapid production of large amounts of a vaccine specific to a new pandemic strain [9,10,12,22,23].

This study showed that H5N1 vaccines mixed with aluminium adjuvant could reach sufficient immunogenicity with a higher dose of vaccine, which is the same as with the non-adjuvant group. However, the AS03 adjuvant consumed as little as 1.9 µg HA antigen and boosted immunogenicity [6,7,8,24]. MF59 improved the body's immune response to AIV with a dose of 3.75 µg but not 1.9 µg of vaccine, according to the EMA criteria [6,24,32].

In addition, this study sought to find evidence regarding the safety of H5N1 vaccines. As shown, there were no serious vaccine-related AEs. All the AEs were mild or moderate reactions. There was no relationship between higher dose and an increased risk of AEs.

The source of the H5N1 virus particles were different in the articles. The particles came from cell cultures, recombinant viral particles or embryo cultures. However, these differences did not affect our conclusions regarding immunogenicity and safety in this study.

The cross-protection effects of the H5N1 vaccine should be assessed in future reports. The adjuvants resulted in less consumption of AIV while inducing a sufficient immune response. The vaccine may have the ability to boost long-term immunogenicity, given that there are no serious adverse reactions and that the vaccine could generate cross-protection.

This study is supported by International Cooperation Project (2011DFR30420); Scientific Research Projects of Health Care (200802023); National 863 Program (2012AA02A404); National Science and Technology Major Project (2013ZX10004003-003-002), Innovation Team in Yunnan Province(2015HC027); Medical Science and Technology Innovation Project of Chinese Academy of Medical Sciences (2016-I2M-3-026).

We confirm that the manuscript has been read and approved by all named authors and that there are no other persons who satisfied the criteria for authorship but are not listed. We further confirm that the order of authors listed in the manuscript has been approved by all of us. We confirm that we have given due consideration to the protection of intellectual property associated with this work and that there are no impediments to publication, including the timing of publication, with respect to intellectual property. In so doing we confirm that we have followed the regulations of our institute concerning intellectual property.